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Oncology
Credited Ph.D. programme
Prof. Dr. András Jeney
Apoptotic and proliferative activity in Neuroblastoma and
PNET, tumors of the Thyroid gland and Parathyroid gland
Dr. Parvaneh Farid
Supervisor: Prof. Dr. Béla Szende
Semmelweis University 1st Department of Pathology and Experimental Cancer Research
Budapest, 2003
2
Introduction 3
Apoptosis in general 5
The TUNEL enzymatic labelling assay & immunohistochemistry 9
Morphology of apoptosis 10
Cell Suicide or cell sacrifice 12
Apoptosis in endocrine cells 13
Role of P53 in apoptosis 13
Bcl-2, an inhibitor of apoptosis 16
Function of Bax 17
Retinoic Acid Receptor 18
Neuroblastoma 20
Classification of Neuroblastomas 21
Primitive Neuroectodermal Tumors-PNET 21
Retinoids and apoptosis in Neuroblastoma 22
Genetic heterogeneity of Neuroblastoma 23
Apoptosis and response of Neuroblastoma to chemotherapy 24
Spontaneous apoptosis & RAR incidence in Neuroblastomas & PNET 26
Material & methods 26
Results 29
Discussion 31
Tumors in thyroid glands 33
Apoptosis in Thyroid tumors 35
Materials & methods 36
Results 38
Discussion 39
Our contribution to WHO 42
Tumors of the parathyroid glands 44
Apoptosis in Parathyroid tumors 46
Materials & methods 47
Results 48
Discussion 58
Conclusion 60
Abbreviations 62
References 64
3
Introduction
A noble man of Persia by the name Baha’u’llah (1817-1892) once explained: ”the
stages that mark the wayfarer’s journey from the abode of dust to the heavenly
homeland are said to be seven, the first is the Valley of Search, the steed of this valley is
patience”. This is a traveller’s tale. It has been hoped to tread the path of a real traveller,
passing through the valleys of love, knowledge, unity, contentment, and wonderment,
arriving to the seventh valley, which would lead us to a result in our research.
Over the past two decades there has been an increase in the findings on apoptosis. More
than 30 new molecules have been discovered, whose known functions have exclusively
to do with the initiation or regulation of apoptosis. A further twenty molecules at least,
although already associated with important roles in signalling or DNA replication,
transcription or repair, have been recognised as affecting the regulation of apoptosis.
This remarkable process is responsible for cell death in development, normal tissue
turnover, atrophy induced by endocrine and other stimuli, negative selection in the
immune system, and a substantial proportion of T-cell killing. It also accounts for many
cell deaths following exposure to cytotoxic compounds, hypoxia or viral infection. It is
a major factor in the cell kinetics of tumors, both growing and regressing (1). Many
cancer therapeutic agents exert their effects through initiation of apoptosis, and even the
process of carcinogenesis itself seems sometimes to depend upon a selective, critical
failure of apoptosis that permits the survival of cells after mutagenic DNA damage. It
has been recognized that most if not all physiologic cell death occurs by apoptosis and
that alteration of apoptosis may result in a variety of malignant disorders (2).
Consequently in the last years interest in apoptosis has increased greatly. Great progress
has been made in the understanding of the basic mechanisms of apoptosis and the gene
products involved (3,4).
The aim of our study has been to learn more about the apoptotic and proliferative
activities in certain endocrine cells, namely Thyroid, Parathyroid tumor cells,
Neuroblastoma and some Primary Neuroectodermal Tumors (PNET) as they crossed
our path with their rarity. The value of these parameters was studied regarding the
prognostic and predictive values and their role in differential diagnosis. Having
examined P53, Bcl-2 and Bax and RAR in different tumors of the tissues mentioned
above, new dimension has been observed. Although few apoptotic tumor cells could be
detected in our study in any of the benign or malignant tumors, this may be relevant to
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the high expression of Bcl-2, since Bcl-2 is known to secure cell survival (5). The
expression of Bcl-2, the qualitative and quantitative differences in different tumors of
thyroid glands, be it in the adenomas or follicular adenocarcinomas, compared to its
expression in papillary carcinomas had been observed to vary greatly, worth noticing
while doing differential diagnosis in practice. The level of Bcl-2 expression in tumors
was related to the apoptotic index as measured by morphologic assessments and
terminal deoxynucleotidal transferase-mediated dUTP-biotin nick end labelling
(TUNEL) assays of DNA fragmentation (6). The high expression of Bcl-2 in adenomas
of the thyroid gland suggests the susceptibility to transformation to malignancy. With
the knowledge that Bcl-2 inhibits apoptosis in a number of different cells, it becomes
clear that prolonged survival of cells overexpressing Bcl-2 is a factor in predisposition
to malignancy (5). From the practical point of view, determination of P53, Bcl-2 and
Bax ratio in thyroid tumors may contribute to the differentiation between adenomas and
specially follicular carcinomas (7). In parathyroid glands on the other hand the
qualitative or quantitative difference between apoptotic and mitotic activity as well as
P53, Bcl-2 and Bax expression has been examined when parathyroid adenomas and
hyperplasias were compared. Nuclear positivity observed in some of the adenomas may
be the sign of tendency to malignant transformation. Co-expression of P53, Bax and
Bcl-2 has been examined to find a relation between mitotic and apoptotic activity
hoping to find a good reason for the very low rate of malignancy in the parathyroid
glands.
Neuroblastomas are heterogenous in terms of genetic features and clinical behaviour. A
remarkable feature of these tumors is the propensity to undergo spontaneous
differentiation. Furthermore some tumors are very responsive to chemotherapy, but
others are quite resistant. Therefore the spontaneous apoptosis has been examined.
Certain pharmacological agents with variable mechanisms of action can induce
apoptosis in neuroblastoma calls. Of this group the most thorough understanding has
emerged from studies on the retinoids. All-trans retinoic acid (ATRA) can induce
neuroblastoma cells to differentiate or undergo apoptosis depending on the cell
phenotype (8). Several studies have been dedicated to the effects of retinoids on
neuroblastomas. It has been observed that the effects of the retinoids are agent-specific
and dose-dependent (9). 13-cis retinoic acid is capable of lowing growth and inducing
differentiation, at least in vitro. However its effects on inducing programmed cell death
in neuroblastomas are less clear (8). 9-cis retinoic acid is more effective than ATRA at
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inducing apoptosis in neuroblastoma cells (10). Successful induction of apoptosis by 9-
cis retinoic acid requires that it be cleared from the cells by a washout technique in
culture, which may have implications for in vivo dosing with this agent. In
neuroblastomas the incidence of spontaneous apoptosis as well as its relation to retinoic
acid receptor positivity was examined (11). In cases of PNET, however, the apoptotic
index as well as retinoic acid receptor positivity was quite different from that of
neuroblastoma. The level of Bcl-2 expression in neuroblastomas is inversely related to
the apoptotic index. There have been conflicting reports about the relationship between
Bcl-2 expression and prognostic features associated with this type of tumor when it
comes to the association between Bcl-2 expression and MYCN amplification. The
findings of one group were an association between Bcl-2 expression, Myc N
amplification, and unfavourable histology (12). Other researchers, however, failed to
confirm these correlations (13). In neuroblastoma cell lines, both Bcl-2 and Bcl-Xl (but
not Bcl-Xs) are expressed (14). Bcl-2 is primarily expressed in lines of chromaffin
lineage, Bcl-Xl is expressed in both chromaffin and nonchromaffin lineage
neuroblastoma cell lines (6). Generally neuroblastoma cell lines that express high levels
of one protein express lower level of the other (14).
Apoptosis in general
Like people, cells die in different ways: accident, murder, old age, even suicide. Cells
that are chosen to die either because they are superfluous, diseased, or have served their
useful purpose don’t just fall apart and expire, they go through a predictable, well
choreographed series of events. The cells round up, their outer membranes form bulges
called blebs, nuclear membranes and some internal structures break down, the nuclear
DNA is fragmented by enzymes, and finally the cell breaks into pieces that are
devoured by still vital neighboring cells. This mode of cell death, in which single cells
are deleted in the midst of living tissues, is called ‘apoptosis’. Apoptosis is a well
defined morphological phenomenon. Eventually, the cell breaks into small membrane-
surrounded fragments (apoptotic bodies), which are cleared by phagocytosis without
inciting an inflammatory response. The release of apoptotic bodies is what inspired the
term “ apoptosis “ from the Greek, meaning “to fall away from” and conjuring notions
of the falling of leaves in the autumn from deciduous trees (1).
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Cell death can occur by either of two distinct mechanisms, necrosis or apoptosis
(15,16). In addition, certain chemical compounds and cells are said to be cytotoxic to
the cell, that is, to cause its death. The question here can arise of what’s the difference
between these terms? To clear up these two terminologies, some basic definition will
follow. Necrosis or accidental cell death is the pathological process that occurs when
cells are exposed to a serious physical or chemical insult. Apoptosis (normal or
programmed cell death) is the physiological process by which unwanted or useless cells
are eliminated during development and other normal biological processes. Cytotoxicity
is the cell-killing property of a chemical compound such as food, cosmetic or
pharmaceutical or a mediator cell (cytotoxic T-cell). In contrast to necrosis and
apoptosis, the term cytotoxicity does not indicate a specific cellular death mechanism.
For example, cell mediated cytotoxicity (that is cell death mediated by either cytotoxic
T lymphocytes (CTL) or natural killer (NK) cells combines some aspects of both
necrosis and apoptosis (17,18). There are many observable morphological and
biochemical differences between necrosis and apoptosis (16). Necrosis occurs when
cells are exposed to extreme variance from physiological conditions e.g. hypothermia
and hypoxia which may result in damage to the plasma membrane. Under physiological
condition direct damage to the plasma membrane is evoked by agents like complement
and lytic viruses. Necrosis begins with an impairment of the cell ability to maintain
homeostasis, leading to an influx of water and extracellular ions. Intracellular
organelles, most notably the mitochondria and the entire cell, swell and rupture (cell
lysis). Due to the ultimate breakdown of the plasma membrane, the cytoplasmic content
including lysosomal enzymes are released into the extracellular fluid. Therefore in vivo
necrotic cell death is often associated with extensive tissue damage resulting in an
intense inflammatory response (17). Apoptosis in contrast, is a mode of cell death that
occurs under normal physiological conditions and the cell is an active participant in its
own suicide. It is most often found during normal cell turnover and tissue homeostasis,
embryogenesis, induction and maintenance of immune tolerance, development of the
nervous system and endocrine dependent tissue atrophy. Cells underlying apoptosis
show characteristic morphological and biochemical features (18). These features include
chromatin aggregation, nuclear and cytoplasmic condensation, partition of cytoplasm
and nucleus into membrane bound vesicles (apoptotic body) that contain ribosomes,
morphologically intact mitochondria and nuclear material. In vivo, these apoptotic
bodies are rapidly recognized and phagocytized by either macrophages or adjacent
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epithelial cells (19). Due to this efficient mechanism for the removal of apoptotic cells
in vivo no inflammatory response is elicited. In vitro, the apoptotic bodies as well as the
remaining cell fragments ultimately swell and finally lyse. This terminal phase of in
vitro cell death has been termed secondary necrosis.
Apoptosis or programmed cell death (PCD) is a genetically controlled process whereby
cells die in response to environmental or developmental cues. The morphological
characteristics of apoptosis include cytoplasmic blebbing, chromatin condensation and
nucleosomal fragmentation (2). Apoptosis is relevant to a range of biological processes,
including differentiation, development, cell maturation and immunologic function
(3,20). Dead cells are rapidly phagocytized to prevent damage to neighboring cells. Too
much cell death may produce neurodegenerative diseases and impaired development,
while insufficient cell death can lead to increased susceptibility to cancer and sustained
viral infection. Progress has been made in the past decade to identify many of the basic
components that contribute to apoptosis, including transcriptional mediators, membrane
bound receptors, Bcl-2 family members, kinases/phosphatase, and cysteine proteases.
Bcl-2 was one of the first genes shown to regulate apoptosis (21) and can inhibit
apoptosis in a wide variety of systems. Bcl-2 belongs to a growing family of genes that
can either positively or negatively regulate apoptosis. One of these gene products, Bax,
binds to Bcl-2 and antagonizes its ability to block apoptosis (22). Another critical
element of the apoptotic process is the activation of cystein protease, which is currently
referred to as caspases. In general, the caspases act downstream of Bcl-2-like proteins to
induce apoptosis. Thus the regulation of apoptosis appears to be a precarious balance
between factors that promote survival and those responsible for initiating and executing
cellular suicide or maybe sacrifice.
What causes these morphological changes that we recognize as apoptosis and the
biochemical changes often associated with this phenomenon? The answer is proteases,
especially activation of a family of intercellular cystein proteases which cleave their
substrates at aspartic acid residue, known as caspases for Cysteine Aspartyl-specific
proteases. The observation that caspases cleave their substrates at Asp residues and are
also activated by proteolytic processing at Asp residue makes evident that these
proteases collaborate in proteolytic cascades, whereby caspases activate themselves and
each other (23). Mammalian caspases appear to constitute an autolytic cascade, some
members (notably caspase 8 or FLICE) being “apical” and more susceptible to
modification by endogenous regulatory proteins, while others (notably capase 3 also
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called CPP32, Yama and apopain) enact the final, irreversible commitment to death.
Study of caspase substrates is providing interesting insights into the ways in which cells
dismantle their structure and function. Such substrates include cytoskeleton proteins
such as actin and fodrin and the nuclear lamins, but also an array of regulatory and
chaperone like proteins whose function is altered by cleavage in subtle and suggestive
ways (24). Caspases appear to be in most if not all cells in inactive proenzyme form,
awaiting activation by cleavage. One of the killing mechanisms of cytotoxic T cells is a
protease, granzyme B, that is delivered to the target cell by the T cell granules and
triggers these latent pro-enzyme. There are endogenous triggers also, and the first to be
discovered, the C. elegans CED4 protein and its mammalian homologue, is particularly
intriguing because of its mitochondrial origin. The CED4 could be the signal that
initiates apoptosis under conditions of shut down of cellular energy metabolism, or
when there is a critical level of cell injury affecting mitochondrial respiration. In this
way CED4 may act as the link between agents long known to be associated with
mitochondrial injury, such as calcium and reactive oxygen species, and the initiation of
apoptosis. A second mitochondrial protein of great significance in apoptosis is BCL-2, a
mammalian homologue of nematode CED9 protein. Bcl-2 has the tertiary structure of a
bacterial pro-forming protein, and inserts into the outer membrane of mitochondria. It
abrogates apoptosis, probably through binding CED4 and another protein Bax, with
which it forms heterodimers and which, like CED4, is also a “ killer ” protein (22). Both
Bcl-2 and Bax have several structurally and functionally similar homologues and some
of this family at least also tap into other cell membranes such as the outer nuclear
membrane and the endoplasmic reticulum.
So are there other sources of death transducers, activating the caspase cascade
because of injury to or signals arising in other parts of the cell than mitochondria? There
are already examples that show that the answer is yes. Thus, the onco-suppressor
protein P53 is activated following some types of DNA damage and can trigger
apoptosis. One way whereby this can happen is through transcriptional activation of
Bax7. The second messenger ceramide, a product of membrane linked acid
sphingomyelinase activation, may act as a signal for plasma membrane damage (25).
And a powerful caspase activating system is mediated by cytokine receptors of the
tumor necrosis factor family, notably fas/apo-1/CD95, TNF receptor I, and others.
These mechanisms for coupling cell injury to apoptosis have mostly depended on
activation of pre-formed proteins. Apoptosis can also be initiated by transcriptional
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mechanisms, although rather little is known about most of them. An outstanding
example is Drosophila gene reaper, transcriptionally activated around two hours prior to
developemental and injury- induced deaths in this organism. Drosophila apoptosis can
occur without reaper transactivation but requires very substantially enhanced stimuli,
suggesting that reaper adjusts a threshold for apoptosis initiation (26). Another gene
whose transcription can initiate death is the familiar immediate early gene c-myc (27).
Transcriptional activation of c-myc initiates entry into DNA synthesis and is required
for sustained re-entry in repeated cell cycles, but c-myc activation in the absence of
concurrent cytokine support triggers apoptosis. This can also be interpreted as a
“threshold regulatory” effect; c-myc expression increases the cellular requirement for
survival factors such as IGF-1. Impressive confirmation of the significance of these
pathways to apoptosis is available from study of trans forming viruses. These are
hardened survivors in the labyrinth of cell regulation, and have found keys to allow
escape from cell death in a variety of ways. Thus the transforming papovavirus SV40,
adenovirus type 12, Human Papilloma Virus type 16 and Epstien-Barr Virus all have
proteins that inactivate apoptois through inactivation of P53 or binding of Bax (28).
There are transcriptional and non-transcriptional pathways for activation of
apoptosis, and they play through common effector events mediated by caspases and
regulated by members of the Bcl-2 family. Underlying this simple scheme, however, is
an extraordinary complexity. Thus inactivation of Fas signalling appears to neutralize
the ability of both c-myc and P53 to initiate apoptosis (29). Many of the proteins
mentioned above have alternative splice variants that have apposite effect. And we still
have little idea of the relevance of intracellular location or of the cell lineage to the
activity of most of the apoptosis proteins. Many other gene products, including
oncoproteins such as RAS and ABL, can influence susceptibility to apoptosis but in
some cases a single oncoprotein may either increase or decrease susceptibility
depending on the context. Perhaps it is not surprising that a cellular function as
important and irreversible as death should be subject to a huge range of course and fine
controls.
The TUNEL enzymatic labelling assay
One of the methods for studying apoptosis in individual cells
There are different methods by which apoptosis could be detected in individual cells,
enzymatic labelling and staining with fluorochrome. In our study we have used
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enzymatic labelling, the so-called TUNEL enzymatic labelling assay. Extensive DNA
degradation is a characteristic event, which often occurs, in the early stage of apoptosis.
Cleavage of the DNA may yield double-stranded, LMW DNA fragments (mono-and
oligonucleosomes) as well as single strand breaks (nicks) in HMW-DNA. Those DNA
strand breaks can be detected by enzymatic labelling of the free 3’-OH termini with
modified nucleotides (X-dUTP, X= biotin, DIG or fluorescein). Suitable labelling
enzymes include DNA polymerase (nick translation) and terminal deoxynucleotidyl
transferase (end labelling). DNA polymerase I catalyze the template dependent addition
of nucleotides when one strand of a double-stranded DNA molecule is nicked.
Theoretically, this reaction (In Situ Nick Translation, ISNT) should detect not only
apoptotic DNA, but also the random fragmentation of DNA by multiple endonucleases
occurring in cellular necrosis. Terminal deoxynucleotidyl transferase (TdT) is able to
label blunt ends of double-stranded DNA break independent of a template. The end-
labeling method has also been termed TUNEL (TdT-mediated X-dUTP nick end
labelling). The TUNEL method is more sensitive and faster than the ISNT method. In
addition, in early stages cells undergoing apoptosis were preferentially labelled by the
TUNEL reaction, whereas necrotic cells were also identified by in situ nick translation
(ISNT). The in situ nick translation (ISNT) is template dependent and TUNEL is
template independent; experiments suggest the TUNEL reaction is more specific for
apoptosis and the combined use of the TUNEL and ISNT is helpful to differentiate
cellular apoptosis and necrosis (30).
Morphology of apoptosis Apoptosis may be divided into three distinct stages: Commitment, execution and
clearance. It is becoming increasingly apparent that a variety of factors influence a cell’s
commitment to the death program in response to an apoptotic stimulus. These include
the cell’s metabolic state, the extent and type of damage induced by the agent, the cell’s
genotype, and the relative expression of growth,- survival-, and death-promoting
factors. Disturbances in any one of these factors may affect a cell’s susceptibility to
apoptotic stimuli, resulting in an inappropriate cellular fate, possibly having significant
consequences on tissue development or disease progression. Altered susceptibility to
apoptosis is particularly relevant to tumorigenesis and tumor progression and for the
development of effective cancer treatment strategies. In the stage of commitment the
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cell having received a potentially lethal apoptotic stimulus, becomes irreversibly
commited to death. It is in the execution stage where major structural changes occur.
Clearance is when cellular remnants are removed by phagocytosis. The structural
changes that occur during the execution phase were described by Kerr et al (1) and are
now becoming better understood mechanistically. During the execution phase,
coordinated morphological and biochemical changes occur within the nucleus,
cytoplasm, organelles, and plasma membrane. The most easily recognizable features of
these events are changes that occur within the nucleus. Chromatin condenses and
aggregates along the nuclear periphery in a crescent shaped pattern. Molecular
characterisation of the chromatin reveals an ordered degradation of the DNA by a
cation-dependent endogenous nuclease, first into large fragments and finally into
nucleosomal fragments. However, cleavage of chromatin to nucleosomal fragments
does not occur in all cell types and can be inhibited without blocking the other changes
of apoptosis (31,32). Parallel with chromatin condensation, the nuclear ultrastructure is
changed. The nuclear lamina, an intermediate filament network that maintains nuclear
envelope integrity and nuclear pore distribution, is proteolytically cleaved (33,34).
Disruption of the nuclear structural framework may then allow nuclear pore clustering
(35) and fragmentation of the nucleus into chromatin-containing fragments, many of
which retain a trace of the nuclear membrane. Other cytoplasmic organelles remain
structurally intact, although mitochondrial dysfunction is associated with apoptosis (36).
Reduction in the mitochondrial transmembrane potential, uncoupling of electron
transport from ATP synthesis, and increased generation of reactive oxygen species
precede the nuclear changes. In the cytoplasm, protein cross- linking occurs through the
action of transglutaminase (37), cytoskeletal filaments aggregate in parallel arrays, and
the endoplasmic reticulum dilates and fuses with the plasma membrane, creating pock-
like craters at the point of fusion. The structural integrity of the plasma membrane is
further compromised by the loss of membrane phospholipid asymmetry, microvilli, and
cell-cell junctions. The cell rounds up, dissociates itself from its neighbours, and shrinks
dramatically, throwing out protuberances that separate into membrane bound ‘apoptotic
bodies’. Within tissues, apoptotic cells and apoptotic bodies are recognized and rapidly
phagocytosed by neighboring cells or macrophages. Once the execution phase of
apoptosis is activated, the entire process proceeds rapidly, and is completed within a
few hours (38). The duration of this phase is relatively invariant with respect to cell type
and apoptotic stimulus, suggesting that the final stage of apoptosis proceed through a
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common pathway. However, the commitment phase, the time from reception of the
apoptotic stimulus until irreversible initiation of the execution phase is extremely
variable, being dependent on cell type, apoptotic stimulus position within the cell cycle,
and expression of various death modulating factors. Many of the factors that influence
commitment or cellular susceptibility to apoptosis, are involved directly in the reception
and transduction of the apoptotic signal. Strikingly most of the genes involved in the
regulation of apoptosis that have been identified during the past few years have turned
out to be also of importance in carcinogenesis.
Cell suicide or cell sacrifice
Apoptosis, or programmed cell death, is a critical process in many areas of biology such
as morphological modelling of tissues during development, removal of autoreactive
immune cells, hormonal or age-related tissue atrophy and normal cell turnover. During
apoptosis, cells, by virtue of their own genetic makeup, die and are deleted for the
overall good of the organism (39). It is also said in literature that cells in multicellular
organisms can kill themselves by activating a suicidal genetic program in response to a
wide variety of signals, including hormones, cytokines, ionising radiation, and
chemotherapeutic agents and that this process of cell suicide is called apoptosis which
usually occurs under physiological conditions (40). The phenomenon of apoptosis has
long been known as programmed cell death, which is fundamental for embryonic
development, such as in metamorphosis, morphogenesis and synaptogenesis (41). While
apoptosis is energy requiring process, functional mitochondria are necessary to provide
adequate energy for the process to reach completion, so it might not be quite fair to call
the process a suicidal process, with a negative connotation of lacking energy and will
power. If cells die because of a signal, be it cytokines or chemotherapy, then they have
been killed and murdered. On the other hand if there is a process where cells die by
virtue of their own genetic make up for the overall good of the organism then this is a
sacrifice a cell makes, since sacrifice involves renouncing that which is lower for that
which is higher. This happens during the embryological development, when a cell
which can not serve the purpose anymore dies and is deleted from the environment. A
tree which bears no fruit is good for fire. It is worthwhile reviewing the terminologies
that have been given to certain processes in the life of a cell.
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Apoptosis in endocrine cells
Because of the fine balance that is maintained between cell proliferation and
death, there is little change in the overall number of cells during the adult life of
multicellular organisms. Cell generation occurs via mitosis, a process that is relatively
well understood compared to the process by which cell die. With regard to cell death,
there are two distinct mechanisms by which this process can occur: necrosis and
apoptosis. Necrotic cell death is characterized by cell swelling and lysis, with the cell
playing no active part in the process. It is also associated with a characteristic
inflammatory response. Apoptosis, in contrast, is a genetically controlled process and is
probably the counterbalancing force to mitosis under normal physiological conditions.
In contrast to mitosis, very little is known about how apoptosis is regulated, but it is
known that the cell plays an active part in its own demise. A variety of genes that play
regulatory role in apoptosis have been identified. These fall into two distinct groups,
those that drive the process and those that inhibit it. In the former category are genes
such as myc and P53 and in the latter genes such as Bcl-2 and Bcr-abl. What is striking
about this observation is that many of them are oncogenes and have been implicated in
the development of numerous cancers. Bcl-2, Bax and P53 expression has been
examined by us (42) in different endocrine cells as factors influencing apoptosis by
either suppressing, inhibiting or causing apoptosis. Our study has been aiming to help
the differential diagnosis in the case of thyroid and parathyroid tumors and in giving
weigh to predictive values. It is important to ponder upon the role and function of these
proteins, finding out how and in what way they act in the process.
Role of P53 in apoptosis
The tumor suppressor gene P53 is the most commonly mutant gene in human cancers
(43). P53 protein levels rise in response to genotoxic damage. This rise in p53, which
results from increased transcription and protein stabilization, is responsible for initiating
G1/S-phase cell cycle arrest, DNA repair and /or apoptosis. Because p53 is a principal
mediator of the cellular response to genotoxic damage, its altered expression and
function may allow the propagation of potentially harmful genetic lesions leading to
oncogenesis. The first clear demonstration that p53 expression could induce apoptosis
14
was based on a cultured cell line transfected with a temprature-sensitive p53 mutant
(44). P53 can induce apoptosis in different ways 1)by activating bax, a proapoptotic
member of bcl-2 family, 2) by enhancing the expression of killer receptor, 3) by
preventing such genes transcription which their products inhibit p53-dependent
apoptosis, 4) by protein-protein mutual effect e.g. it binds to transcription/ repair
complex (45). In 1993 concurrent studies by Lowe et al (46) and Clarcke et al (47)
demonstrated that p53 was involved in the cellular apoptotic response to genotoxic
damage. In these studies, thymocytes from mice rendered homozygous null for p53 by
germline targeting were found to be completely resistant to the apoptosis seen in wild
type cells shortly after exposure to ionising radiation or chemical induced DNA double-
strand breaks (46,47). However, p53 genotype had no effect on glucocorticoid- induced
apoptosis in these cells. These findings established the existence of distinct p53-
dependent and p53 independent mechanisms of apoptosis. Moreover, because p53-null
mice showed a high incidence of thymic lymphomas, the results gave some support to
the hypothesis that p53 mutations may facilitate tumor initiation and progression by
producing defects in apoptosis. This hypothesis has been further supported by elegant in
vivo experiments using transgenic mice engineered to selectively express SV40 large T
antigen product, which can selectively inactivate Rb or p53, in choroid plexus cells. In
the animals expressing the full length SV40 large T transgene, which inactivates both
Rb and p53, aggressive choroid plexus tumor formation was observed. In contrast,
tumor growth occurred at a considerably slower rate in animals expressing a truncated
SV40 large T transgene capable of inactivating Rb but not p53 (48). Analysis of these
tumors revealed that p53 genotype had no appreciable effect on rates of proliferation.
Instead, rates of apoptosis were low in those tumors in which p53 was inactivated,
suggesting that p53 associated tumor suppression due to enhanced rate of apoptosis
rather than decreased rate of proliferation. Similar results have been reported in
additional tissue-specific transgenic system that addressed the role of p53 and Rb in
tumor development (49,50).
Gene expression has been shown to be required for both apoptosis and survival
depending on the cell type and stimulus. Indeed, inhibition of RNA and protein
synthesis can block apoptosis induced by a number of circumstances. Others have
shown that in some cases RNA and protein synthesis inhibitors either do not block or
actually promote cellular demise (51). The basic machinery for apoptosis is
constitutively expressed but can be modulated by changes in Gene expression in order
15
to trigger the death program. p53 was first detected in rodent cells transformed by
simian virus SV40 in a complex with the transforming protein SV40 T antigen,
suggesting that it plays a role in growth control (52). Subsequently, it was noted that
p53 mutations occur in a wide variety of tumors including lung, breast, colon,
esophagus, liver, bladder, ovary, brain and haemopoetic tissues (43). In fact, p53 loss-
of- function mutations are the most common genetic alteration found in human cancer.
Furthermore, overexpression of wild-type p53 can suppress tumor formation in culture
(53,54,55). These data suggest that p53 function as a tumor suppressor gene and has
sparked extensive research into understanding its mechanism of action. p53- mediated
transrepression, on the other hand, occurs on genes lacking the p53 DNA binding site
and is probably dependent on interactions with components of the basal transcriptional
machinery (56,57). Mutational analysis of p53 has revealed distinct domains that
contribute to its gene regulatory activity. Consistent with its role as a tumor suppressor
gene, expression of p53 can induce either cell cycle arrest in G1 or apoptosis (58,44).
Functional p53 is required for apoptosis induced by ionizing radiation and
chemotherapeutic drugs (59,47) as well as transforming oncogenes like c-myc (60,61).
The function of p53 is likely to depend on the cell type and/or physiological
circumstances. Indeed, cytokines (44) and growth factors can affect the ability of p53 to
induce apoptosis or growth arrest. These results have led to the hypothesis that
genotoxic induction of p53 acts as a checkpoint control to stop further progression in the
cell cycle and thereby maintain genomic integrity (62). The mechanism by which p53
induces apoptosis, however, is somewhat unclear. Generally, p53 mediated apoptosis is
dependent on its gene regularity capability. Another critical issue that remains is
whether p53-dependent cell death involves activation or repression of cellular genes.
Studies have indicated that at least in some cases p53 induced apoptosis is not affected
by the presence of RNA and protein synthesis inhibitors, suggesting that repression
rather than activation is the primary mechanism (63). Several p53 responsible genes
have been identified that could affect cell survival, including bcl-2 (64). Concerning
thyroid neoplasms there exists various reports on the overexpression of p53 especially
in poorly and undifferentiated carcinomas (65,66).
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Bcl-2, an inhibitor of apoptosis
One of the first mammalian genes discovered to regulate cell death was the anti-
apoptotic gene bcl-2 (67). The bcl-2 proto-oncogene was originally discovered as a
common translocation in non-Hodgkins B-cell lymphoma (68,69). This chromosomal
translocation event places the bcl-2 gene under the transcriptional control of the
powerful enhancer elements of the immunoglobulin heavy chain resulting in high levels
of bcl-2 expression and the abrogation of normal programmed cell death and promotion
of malignant potential. Disruption of bcl-2 in “ knock out “ mice leads to impaired
kidney function manifested by polycystic disease and postnatal immune failure due to
dramatic cell loss through apoptosis. Thus, gain of bcl-2 function is associated with
tumor development (68,69,70,71,72), while loss of bcl-2 has only restricted
consequences to normal development. This suggests that there may be some redundancy
in the bcl-2 family or that other members have a more critical role. Several bcl-2 like
genes have been identified that can promote, rather than inhibit, apoptosis. The best-
characterized and prototypic gene of this class is bax (22). While overexpression of bax
can induce apoptosis, disruption of this gene in “ knock out “ mice leads to lymphoid
hyperplasia (73). Recent data also demonstrates that expression of bax can suppress
tumorigenesis in vivo (74). These data suggest that bax plays a critical role during
apoptosis. Biochemical studies have indicated that bax directly interacts with itself as
well as several anti-apoptotic proteins such as bcl-2 (22). Interaction between death
promoting and suppressing bcl-2- like proteins has lead to a model for explaining how
these proteins function. According to this model, the ratio between survival factors,
such as bcl-2, and death promoters, such as bax, controls the fate of the cell. However,
the biochemical mechanism of these proteins is still unclear. What is also unclear is
which set of bcl-2 proteins act as effector molecules. For example, do the death
promoting genes simply inhibit a required survival factor (e.g. bcl-2, bcl-xl ) or do they
actually trigger apoptosis?
Bcl-2 and its family members bind to a number of unrelated proteins that has provided
insight into their apoptotic activity. Indeed, bcl-2 related proteins are generally found
associated with membrane structures, particularly the mitochondria, endoplasmic
reticulum, and the nuclear envelope. Furthermore, in vitro experiments demonstrate that
bcl-xl can facilitate ion transport across a lipid bilayer. It will be of interest to determine
if the bcl-2 family members actually function as channels in vivo and if this activity is
17
responsible for apoptosis regulation. These and other studies have lead to several
hypotheses regarding the biochemical mechanism of this family of proteins. One
possibility that has emerged is that they form a channel capable of regulating ion flux,
such as calcium. Calcium is a critical component of signaling pathways and high level
of intracellular calcium is often associated with cell death, bcl-2 can suppress calcium
release from the endoplasmic reticulum, although it is possible that this is simply a
downstream consequence of its ability to inhibit apoptosis (75). Nevertheless, the
experiments indicating that the bcl-2- like proteins can form ion channels add some
credence to this model. Others have speculated that bcl-2 regulates the generation of
reactive oxygen radicals since bcl-2 is able to attenuate cell death induced by oxidative
damaging agents. (75). Free radicals can contribute to cell death by disrupting DNA,
proteins, and lipids. The localization of bcl-2 at mitochondrial membranes makes it
plausible that the protein is directly involved in oxidative phosphorylation (67).
However further evidence demonstrates that bcl-2 can inhibit apoptosis in cells that lack
mitochondrial DNA and thereby respiration (76). An alternative mechanism for bcl-2
action at the mitochondria may be the control of cctochrom C release. Release of
cytochrome c from the mitochondria into the cytosol is required for caspase activation
and DNA fragmentation in cell free extracts and bcl-2 can act to suppress cytochrom c
efflux from mitochondria (77). It will certainly be important to determine if efflux of
proteins, such as cytochrom C, from the mitochondria actually contributes to apoptosis
in vivo or if this is simply a downstream phenomenon. Additionally, there is tremendous
interest in elucidating the down stream events of apoptosis, most notably caspase
activation.
As to the expression of bcl-2 in endocrine cells, bcl-2 has been reported to be retained in
well & poorly differentiated thyroid carcinomas but absent in undifferentiated thyroid
carcinomas (159,41). Several studies have found that bcl-2 is widely expressed in
neuroblastoma tumors and tumor derived cell lines (13,54).
Function of Bax
Bax has been shown by a number of investigators to be capable of forming either
homodimers with bcl-2 and bcl-Xl by immunoprecipitation and yeast two hybrid select
systems (40,41). Bases on the tendency of bax to promote cell death, it has been
proposed that the relative ratio of bax heterodimers to bax homodimers serves as a
18
switch that dictates the cell fate (78,79). According to this model, exposure of the cell to
cytotoxic insults may promote the formation of bax homodimers. However this death
initiation step may be blocked, if sufficient quantities of the prosurvival factors bcl-2 or
bcl-Xl are present to form heterodimers with bax. Thus depending on the relative ratio
of bax heterodimers to bax homodimers, the cell may proceed toward either cell
survival or cell death. bax dependent cell killing appears to be linked to caspase
activation (80,81). Several reports have suggested that overexpression of bax leads to
the loss of inner mitochondrial potential, a phenomenon also known as mitochondrial
permeability transition (82,83). Observations taken together have led to the proposal
that bax may form pores on mitochondrial surface, leading to the dissipation of
mitochondrial potential and to the release of cytochrome C (84). One could envision
that these series of processes would first require a signalling mechanism that switches
bax from its normal physiologically inactive state, possibly represented by its cytosolic
form, to an active apoptotic state possibly represented by its mitochondria-bound form.
Studying the expression of bax has not been given too much attention in endocrine cells.
Nevertheless there have been studies that bax expression has been negative in normal
thyroid tissue and weakly positive in meudullary thyroid tumor (85).
Retinoic Acid Receptor
Retinoids have profound effects on normal vertebrate development and the maintenance
of normal cellular functions in some adult tissues (86). A group of pharmacological
agents with different mechanism of action can induce apoptosis in neuroblastoma cells
(8). Some examples are retinoids. All-trans retinoic acid (ATRA) can induce
nueroblastic cells to undergo apoptosis. It is therefore not out of place to expand on
Retinoic Acid Receptor (RAR) since we have examined its expression in
neuroblastomas and PNET and its relation to apoptosis (11).
The widespread cellular effects of retinoids are mediated by two classes of nuclear
receptor: the retinoic acid receptors (RAR) and the retinoid X receptors (RXR) (87).
Discovery of the nuclear retinoic acid receptors (RARs) and nuclear retinoid X
receptors (RXRs), has significantly advanced our understanding of the biological basis
of retinoic acid (RA) function. These receptors, which belong to the multigene family of
steroid/thyroid hormone receptors RA-inducible transcriptional regulatory factors
transduce the RA signal at the level of gene expression. There are 3 subtypes (? , ? and
19
? ) of both RARs, and RXRs, each coded for by separate genes. Nuclear retinoid
receptors are ligand-dependent transcription factors which bind specific DNA
sequences, known as retinoic acid response elements (RARE) in the promoter regions of
target genes. RAR-RXR heterodimers, rather than homodimers of individual receptors,
bind with greatest affinity in vitro to RAREs. Single and combination gene knockout
studies in mice, for each of the nuclear retinoid receptors, have demonstrated that
specific receptor subtypes have signaling roles in certain embryonal tissues, however,
functional redundancy also exists between receptor subtypes in other tissues (88). A
large number of tumor cell lines from different tissue origins undergo growth arrest and,
in some cases, differentiation, following retinoid treatment in vitro (88). These
observations have raised the possibility that disordered retinoid signaling may
contribute to some tumorigenic processes, and, moreover, that retinoids may have
therapeutic or chemo-preventive roles in certain human cancers. Abnormal function or
reduced expression of specific RAR, but not RXR, subtypes contribute to the malignant
phenotype in several human cancers (89,90,91,92,93). Several groups have linked
reduced RAR ? expression to the genesis of human epidermal, lung, and breast cancer
(91,93,94,95).
20
Tumors investigated in our study
1. Neuroblastoma
The neuroblastic tumours, derived from primordial neural crest cells which
ultimately populate the sympathetic ganglia, adrenal medulla and other sites, (96) are an
enigmatic group of neoplasms which have the highest rate of spontaneous regression of
all human malignant neoplasms yet one of the poorest outcomes when occuring as
disseminated disease in children. Neuroblastoma is one of the most common childhood
extracranial solid tumors. Alone, it accounts for about 15% of all childhood cancer
death (97). A great deal is known about the molecular biology and genetics of this
neoplasm. Studies strongly suggest that there are at least two, possibly three,
clinicobiologic subsets of neuroblastoma, ranging from those that rarely kill, however
hopeless the clinical situation may appear to be, to those that rapidly cause death despite
all therapeutic efforts. Furthermore, some tumors are very responsive to chemotherapy
but others are quite resistant. Thirty-five percent of these tumors appear during the first
year of life, 20% during the second year. Altogether, 85 to 90% are found in children
younger than five years of age. Most occur sporadically, but in about 20% of instances
there is strong evidence of a hereditary predisposition.
In childhood, about 25 to 35% of neuroblastomas arise in the adrenal medulla. The
remainder occur anywhere along the sympathetic chain, with the second most common
location being the paravertebral region of the posterior mediastinum. Closely following
is the paravertebral region in the lower abdomen, but tumors may arise in numerous
other sites, including the pelvis and neck and within the brain. By contrast, rare
neuroblastomas in adults are found in the head, neck, and legs. Macroscopically,
neuroblastomas range in size from minute nodules (the “ in situ lesions “) to large
masses more than 1 kg in weight. In situ neuroblastomas are reported to be 40 times
more frequent than overt tumors. The great preponderance of these lesions
spontaneously regress leaving only a focus of fibrosis or calcification in the adult.
Indeed, some clinically overt neuroblastomas have regressed or, alternatively, have
undergone differentiation and maturation into a relatively benign ganglioneuroma.
Some neuroblastomas are sharply demarcated and may appear encapsulated, but others
are far more infiltrative and invade surrounding structures, such as the kidneys, renal
vein, and vena cava, and envelope the aorta.
21
Histologically, there is a considerable range of differentiation among these neoplasms.
Most are composed of small, primitive-appearing cells with dark nuclei, scant
cytoplasm, and poorly defined cell borders growing in solid sheets.
Classification of Neuroblastomas
The International Neuroblastoma Pathology Committee (INPC) has adopted a
prognostic system modeled on one proposed by Shimada et al(98). It is an age- linked
classification dependent on the differentiation grade of the neuroblasts, their cellular
turnover index, and the presence or absence of Schwanian stromal development. Based
on morphologic criteria defined in this article, neuroblastic tumors (NTs) were classified
into four categories and their subtypes:
1. neuroblastoma (Schwannian stroma-poor), undifferentiated, poorly
differentiated, and differentiating;
2. ganglioneuroblastoma, intermixed (Schwannian stroma-rich);
3. ganglioneuroma ( Schwannian stroma-dominant ) maturing and mature;
4. ganglioneuroblastoma, nodular ( composite Schwannian stroma-rich / stroma-
dominant and stroma-poor).
1/a. Primitive Neuroectodermal Tumors (PNET)
Some tumors, although of neuroectodermal origin, express few, if any, of the
phenotypic markers of mature cells of the nervous system and are described as poorly
differentiated, or embryonal, meaning that they retain cellular features of primitive,
undifferentiated cells. The most common is the medulloblastoma, which accounts for
20% of the brain tumors of childhood and occurs in the cerebellum. Other poorly
differentiated tumors occur in other locations, and have been collectively termed
primitive neuroectodermal tumors (PNET). Most classification systems continue to
recognize the medulloblastoma as a distinct clinicopathologic category and
acknowledge the presence of other poorly differentiated tumors that occur
predominantly in children. Primitive neuroectodermal tumor is a highly controversial
designation, which has been applied to neoplasms, primarily of childhood, that are
histologically similar to medullobastoma but occur in locations other than the
22
cerebellum, especially the cerebral hemispheres. Many would prefer not to gather all
these poorly differentiated tumors into a single category until there is a clearer vision of
their biological behavior; others find the designa tion useful to evaluate the efficacy of
chemotherapeutic protocols. More information is needed to settle this issue.
Retinoids and Apoptosis in Neuroblastoma
A group of pharmacological agents with variable mechanisms of action can induce
apoptosis in neuroblastoma cells in vitro. Some examples are retinoids, DNA strand
breaking agents like cisplatin and doxorubicin, protein kinase inhibitors and others. Of
this group the most thorough understanding has emerged from studies on the retinoids.
All-trans retinoic acid (ATRA) can induce neuroblastoma cells to differentiate or
undergo apoptosis, depending on the cell phenotype (8). Three phenotypic variants of
neuroblastoma cells in culture have been described. N-type cells constitute the
neuroblastic phenotype. These cells have neurotic processes and display biochemical
characteristic of neuronal cells. S-type cells are substrate-adherent, epithelial in
phenotype, and display biochemical features of immature Schwann, glial, or
melanocytic cells. The third phenotypic variant is I-type cells, with intermediate
morphology and bio-chemical features of both N-and S-type cells. S-type cells do not
express Bcl-2 and are particularly sensitive to induction of apoptosis by ATRA. This is
in contrast to N-type cells, which express Bcl-2 and are less sensitive to RA-induced
apoptosis (99,100). Neuroblastoma cells that differentiate in response to RA are
resistant to chemotherapy- induced apoptosis. This resistence is associated with
regulation of Bcl-2(100). The effect of the retinoids are agent specific and dose-
dependent. 13-cis RA is capable of slowing growth and inducing differentiation, at last
in vitro. However its effect on inducing programmed cell death (PCD) in
neuroblastomas are less clear (8,9). 9-cis RA is more effective than ATRA at inducing
apoptosis in neuroblastoma cell (10). Successful induction of apoptosis by 9-cis RA
requires that it be cleared from the cells by a washout technique in culture, which may
have implications for in vivo dosing with this agent (10). Finally there is a novel
retinoid called N-(4-hydroxyphenyl) retinamide (fenretinide), which appears to be
particularly potent at inducing apoptosis (101,102). The mechanism through which the
retinoids induce apoptosis has not been clearly defined.
23
Neuroblastoma is an embryonal childhood tumor of neural crest origin with a
unique capacity for spontaneous regression and differentiation, in vivo (103,104,105).
In some older patients the tumor may differentiate into a benign ganglioneuroma.
Unfortunately in the majority of patients, neuroblastomas are metastatic at diagnosis and
most of these children ultimately die of their disease, especially if they are over 1 year
of age at diagnosis (96).
Several studies have found that Bcl-2 is widely expressed in neuroblastoma tumors and
tumor derived cell lines (13). Moreover the level of Bcl-2 expression in tumors is
inversely related to the apoptotic index, as measured by morphologic assessments and
terminal deoxynucleotidal transferase-mediated dUTP-biotin nick end- labelling
(TUNEL) assays of DNA fragmentation (6). There have been conflicting reports about
the relationship between Bcl-2 expression and prognostic features associated with this
disease. One group reported an association between Bcl-2 expression, MycN
amplification, and unfavourable histology (84). Other investigations however have
failed to confirm these correlations (13). In neuroblastoma cell lines, both Bcl-2 and
Bcl-Xl (but not Bcl-Xs) are expressed. Bcl-2 is primarily expressed in lines of
chromaffin lineage, Bcl-Xl is expressed in both chromaffin and nonchromaffin lineage
neuroblastoma cell lines. Generally neuroblastoma cell lines that express high levels of
one protein express lower levels of the other (6).
Retinoid treatment of most neuroblastoma tumor cell lines induces growth arrest and
neuritic differentiation in vitro (106) and, thus provides an excellent model for studying
whether abnormal retinoid signaling is a component of the neuroblastoma tumorigenic
process. Untreated neuroblastoma cells express high levels of RAR-? , low level of
RAR-?, and barely detectable levels of RAR ? , while retinoid treatment induces RAR-
? expression some 40-fold and RAR ? expression by four-fold (107,108,109).
Comparative studies of neuroblastoma cells transfected with each of the 3 RAR subtype
have shown that only RAR-? transfectants demonstrates enhanced retinoid sensitivity
and growth inhibition in the absence of added retinoid (110).
Genetic heterogeneity of neuroblastoma Several genetic features have been found to be characteristic of subsets of
neuroblastomas, including hyperdiploidy with whole chromosome gains, amplification
of the MYCN oncogene, and deletions of the short arm of chromosome 1 (1p)(111).
24
Hyperdiploidity is characteristic of tumors in infants, particularly those who are prone
to undergo spontaneous regression or who respond well to chemotherapy. However, the
most aggressive tumors particularly those who are metastatic in older patients
frequently have MycN amplification and the majority of these also have 1p deletion
(111). Partial trisomy for 17q is also a common genetic change in neuroblastomas and
its presence in the karyotype is generally associated with a worse outcome. Other
deletion and rearrangements are also found such as deletion of 11q or 14q, but so far
these features have not been consistently associated with clinical behaviour.
Apoptosis and response of neuroblastoma to chemotherapy
A variety of DNA damaging agents have been shown to induce apoptosis in
neuroblastoma cells in vitro (112,113). Several stud ies have indicated that
neuroblastoma cells undergo apoptosis in response to DNA damaging agent after arrest
in G2/M of the cell cycle (114). Neuroblastoma cells treated with these agents show
classic morphologic features of apoptosis and evidence of DNA damage. The CD95
system has recently been shown to mediate apoptosis induced by chemotherapy in
neuroblastoma (115). CD95, a member of the TNFR superfamily, triggers cell death in a
variety of cell types. In neuroblastoma cells, doxorubicin, cisplatin, and etoposide
induce expression of CD95, as well as the ligand, CD95L (115). Chemotherapy induced
apoptosis mediated by CD95 in neuroblastoma involves activation of ced-3-like
proteases, resulting in cleavage of PARP an enzyme involved in DNA repair (115).
These results suggest that autocrine or paracrine activation of CD95/CD95L leads to a
death response in neuroblastoma cells after treatment with these specific
chemotherapeutic drugs.
Both Bc-2 and Bcl-Xl can modulate chemotherapy induced apoptosis in neuroblastoma
cells. Single-gene transfection studies have shown that deregulated expression of Bcl-2
or Bcl-Xl in a dose dependent manner can confer resistance to apoptosis induced by
cisplatin or 4-hydroxycyclophosphamide, and delay apoptosis induced by etoposide
(116,112,113).
Not all cytotoxic agents induce apoptosis by engaging CD95. Betulinic acid (BA), a
pentacyclic triterpene, induces apoptosis in a number of cell types, including
neuroblastoma, and this appears to be independent of the Cd95 and P53 pathways (117).
BA-induced apoptosis is associated with increased caspase activity, enzymatic
25
processing of the upstream caspase component, FLICE (caspase-8) and PARP cleavage.
BA-induced apoptosis, however, is independent of CD95/CD95L activation. BA-
induced apoptosis is associated with induction of the apoptosis- facilitating proteins, Bax
and Bcl-Xs. Both Bcl-2 and Bcl-Xl can inhibit apoptosis induced by BA in a manner
similar to its protection from DNA-damaging agents (115).
Caspase activation appears to be the final common pathway for induction of apoptosis
by most agents. Staurosporin, an alkaloid inhibitor of protein kinases, is also able to
induce apoptosis in neuroblastoma cells (118). Staurosporin- induced apoptosis is
associated with proteolytic processing of CPP32 (caspase-3), PARP activation, and
PARP cleavage. In some systems, however, PARP activation is not always associated
with proteolytic processing of PARP. Peroxynitrate (PN), which forms through a
reaction of superoxide with nitrous oxide has recently been shown to induce apoptosis
in the NSC34 neuroblastoma spinal cord cell line (119). In this system, PN-induced
apoptosis was associated with morphologic features of apoptosis and increased PARP
immunoreactivity, but there was no evidence of PARP cleavage. It has been suggested
that PN induce DNA damage that stimulates PARP, which depletes intracellular energy
stores, resulting in cell death (119). This possibility will require further investigation,
however, these results suggest that additional pathways may function to mediate
specific drug- induced apoptosis in neuroblastoma.
Therapeutic strategies based on in vitro studies of neuroblastoma cells have led to
several current clinical trials in patients with neuroblastoma. Most notable are the
protocols utilizing retinoic acid (RA) in the treatment of advanced stage disease, which
are based on in vitro assays indicating RA can inhibit neuroblastoma proliferation. A
phase 1 trial utilizing 13 cis-RA following bone marrow transplantation for stage 4
neuroblastoma has suggested that the drug is well tolerated, with minimal toxicity (120),
and its therapeutic efficacy is currently under investigation.
An improved understanding of the molecular pathways controlling the survival/death
response of neuroblastoma cells should provide insight into mechanism that could
ultimately be targeted for novel therapeutic intervention. In support of this possibility
are studies indicating that neuroblastoma cells, regardless of their Bcl-2 or Bcl-Xl
expression, are sensitive to induction of apoptosis by adenoviral-mediated expression of
the death-facilitating protein Bcl-Xs (116). The possibility of therapeutically engaging
the death pathways directly is intriguing, but will require the development of targeted
gene therapy approaches. Finally, therapeutic interventions aimed at either blocking the
26
Trk ligand-receptor interaction, or selectively inhibiting the Trk family of receptors,
may provide a therapeutic approach that is more effective and less toxic than currently
used chemotherapeutic agents. The blue letters are my own work.
Our studies on apoptosis in Neuroblastoma & PNET
Aim: RAR-ß and apoptosis: therapy with RA
The specimen has been received from the 2nd Department of Pediatrics, Semmelweis
University of Medicine, Budapest, Hungary. The great help and assistance of Prof.Dr.
Dezso Schuler and the co-operation of Dr. Peter Hauser and Dr. Maria Babosa is deeply
appreciated.
Little is known about the spontaneous apoptotic activity in neuroblastomas and PNET.
These tumors have a poor prognosis even when treated with irradiation and/or cytotoxic
drugs (121,122). Recently retinoic acid has been reported to be effective in the therapy
of NB, promoting the differentiation of the tumor cells (123). In vitro studies of NB
cells showed that retinoic acid administration induces apoptosis in cell cultures and this
effect was mediated by a retinoic acid receptor (124). The possibility of inducing
apoptosis by retinoic acid treatment has been studied in vitro (124). Nuclear localization
of the receptor by Western blot has been reported, we investigated the posibility to
localize the receptor by immunohistochemical method within the tumor cells of NB and
PNET.
Materials and methods
Twenty-two cases of NB and four cases of PNET were investigated. In a
retrospective study basic clinical data, such as age, sex, location of the tumor,
chemotherapy and/or surgical removal irradiation and survival were registered (Table
1).
Among the children suffering from NB 14 were males (average age 3.40+0.80)
and 8 were female (average age 3.40+1.5 years). Three boys (2.5 and 10 years old) and
one girl (4 years old) had PNET. Seven neuroblastomas and two PNETS were in Evan’s
stage IV, four neuroblastomas were in Evan’s stage II, one neuroblastoma and one
PNET were in Evan’s stage I,and one PNET was in Evan’s stage II at diagnosis (Table
27
2). The tumor tissues were obtained by surgical excision before treatment from the
primary tumor, and after formalin fixation and paraffin embedding 8um thin sections
were cut.
Patient Sex Age
(Year) Chemotherapy Radio-therapy Location Operation Stage/Evans
1.NB F 0.8 Endoxan + ADR + steroid . OPEC None Mediastinum Part.res. St.IV.
2.NB F 1,2 NB-90 RG-C None Mediastinum Part.res. St.III.
3.NB F 8
Vac+OPEC+OJEC(RETINOID)+ Autotransplantation None
Retro-peritoneum Part.res. StIII.
4.NB F 1 None None R. adrenal gland
Total resection St.I.
5.NB F 6 OPEC/OJEC None Retro-peritoneum None St.III.
6.NB M 4 NB-90-RG-C 32 Gy Retro-peritoneum Inoperable St.IV.
7.NB M 1 OPEC+Newcastle+Autotranspl. None
Retro-peritoneum Inoperable St.III.
8.NB M 4 No clinical data/ foreign person/ None
Retro-peritoneum None St.IV.(?)
9.NB M 4 ALL-BFM-88 None Retro-peritoneum Part.res. St.IV.
10.NB M 6 OPEC 30 Gy R. adrenal gland Part.res. St.IV.
11.NB M 3 OPEC None Pelvic lymph node Part res. St.IV.
12.NB M 5 OPEC None Retro-peritoneum Inoperable St.IV.
13.NB F 6 VP-16+Carboplatin None Retro-peritoneum
Auto.bone.m transpl. St.IV
14.NB F 2 n.d. None Bone marrow Bone m. transplant St.IV.
15.NB M 8 OPEC/OJEC None Bone marrow Bone m. transplant St.IV.
16.NB M 3 n.d. None Retro peritoneum
Bone m. transplant NA
17.NB M 0.3 n.d. None Adrenal gland None NA
18.NB F 0.5 HR-NBL-1/ESIOP None Retro-peritoneum None St.IV
19.NB M 2 n.d. None Adrenal gland None St. I.
20.NB M 3 n.d. None Adrenal gland None St. I.
21.NB M 3 n.d. None Retro-peritoneum None St.IV.
22.NB M 1 n.d. None Mediastinum None NA
23.PNET F 4 OPEC/OJEC None Retro-peritoneum Part.res St.IV.
24.PNET M 2 NB-A-89 None Retro-peritoneum Part.res St.I.
25.PNET M 5 VAIA 90 Gy Right. forearm Part.res. St.IV.
26.PNET M 10 NHL-BFM-95 None Mediastinum Part.res. St.II
n.d. = no data;
Table 1.
28
All sections were examined with HE staining. Neuroblastomas were of the small
round cell type and PNET showed the characteristics described elsewhere (125).
ApopDetek (simply sensitive in situ Detection Kit, Dako, Denmark) was used to show
apoptosis (126).
Table 2.
Patient Diagnosis Sex Age (Year) Apoptosis index (%)
RAR-B index (%) Survival (months)
1. NB F 0.8 4,8 22 5 2. NB F 1,2 1 0 24 3. NB F 8 0 0 >24 4. NB F 1 0 0 11 5. NB F 6 0 0 16 6. NB M 4 2 19 13 7. NB M 1 0 10 20 8. NB M 4 0 9 >24 9. NB M 4 1 3 6 10. NB M 6 3,8 17 23 11. NB M 2,3 3,4 34 17 12. NB M 2,5 0 11 20 13. NB F 6 0 30 n.d. 14. NB F 2 0 10 n.d 15. NB M 8 0 90 n.d 16. NB M 3 0 0 n.d 17. NB M 0,3 0 80 nuclear
20 cytopl. n.d
18. NB F 0,5 0 85 cytopl. n.d 19. NB M 2 3 50 cytopl.memb. n.d 20. NB M 3 0 70 n.d 21. NB M 3 0 50 nuclear n.d 22. NB M 1 0 - n.d 23. PNET F 4 5 43 4 24. PNET M 2 19,5 68 25 25. PNET M 5 6,8 41 42 26. PNET M 10 4,1 16 >20
Sections were treated with proteinase K to make the tissue permeable to the
reagents of the labelling and detection procedure. The apoptosis index in the tumor
tissue was determined by examining 2000 tumor cells. The presence of RAR was
29
examined by immunoperoxidase reaction using RAR-B Ab (BIOMOL, Germany,
cat.No.SA-17). This antibody has been reported to show RAR in Western blot (127).
The sections were treated with proteinase K and, to inactivate endogenous peroxidase,
incubated in methanol and H2O2. Undiluted anti-retinoic acid receptor-B was applied as
primary antibody and was incubated overnight at –4 C. The secondary Ab-mouse
immunoglobulin was used in 1:100 dilution the next day- DAB (diaminobenzidine) was
used as a chromogen and methyl green as a counterstain.
The same procedure was performed with RAR-B diluted in 1:5 and 1:10 to test the
sensitivity of the reaction, since according to our knowledge this is the first attempt to
use immunohistochemistry to show RAR. RAR index in tumor tissue was determined
by examining 2000 tumor cells.
Results The clinical data of our
patients are shown in
table1. With the
exception of two cases
(case 3 and case 6), death
was due to progression of
the tumor 5-42 months
after the diagnosis,
irrespective of
localization, histological
types, or mode of therapy.
Only one case (case 3)
received RA therapy, this
patient is still alive.
Apoptosis was shown in
6 NB tumor samples
(table-2).
The apoptotic index was
zero or 1% in 8 children
Fig. 1. NB case scattered apoptotic cells. ApopDetec staining (x300).
Fig. 2. NB RAR positivity on cell surface. Anti-RAR-beta immuno- peroxidase (x300)
30
and relatively low (2-4%) in the other four cases, while it was higher (4.1-10.5%) in
PNET. The RAR index determined by immunoperoxidase reaction in NB was zero to
3% in five cases and 9-34% in seven children (table-2). Scattered apoptotic cells in a
section of neuroblastoma is shown in Figure 1.
RAR index in PNET was 16-68% in all four cases. As shown in table 2, apoptotic index
and RAR index varied parallel to each other, i.e., when apoptosis was frequent, the
RAR index was also elevated. The correlation coefficent between apoptosis and RAR
index was r = 0.47 according to Pearson-Bravis.
RAR positivity appeared in the nuclei of tumor cells both in NB and PNET when the
antibody was applied undiluted (figures 2 and 3), but if 1:5 or 1:10 dilution was also
applied, cell surface and slight cytoplasmic positivity could be detected in the same cell
(Figure 4, 5), along with nuclear positivity, probably due to better resolution of the cell
organelle. There was no evaluable relationship between apoptotic index, age, and stage.
Fig. 3. RAR positivity in nuclei of NB. Anti-RAR-beta immonoperoxidase (x150)
31
Discussion A variety of tumors have been studied for spontaneous and induced apoptosis.
Inducibility of apoptosis in tumor tissue by various drugs or irradiation is considered as
a possible predictive factor (121,122,128). The spontaneous apoptosis index in NB and
PNET has been found to be very low (129, 130). The possibility of inducing apoptosis
by retinoic acid (RA) treatment was first studied by Smith et al (124) in vitro. In their
experiment RA-induced apoptosis in cultured NB cells was studied and this effect was
linked to RAR. Since RA therapy has been administered in some cases of NB (131,132)
Fig. 4. PNET cytoplasmic RAR positivity. Anti-RAR-beta immunoperoxidase (x300)
Fig. 5. NB case Tumor cell nuclei are strogly RAR positive, anti-RAR-beta immunoperoxidase (x300).
32
detection of RAR may predict the effectiveness of RA treatment. Although nuclear
localization of the receptor by western blot (133) has been reported we demonstrated by
immunoperoxidase reaction that at least a certain proportion of the receptor may be
situated on cytoplasmic membranes.
As for NBs, not all were RAR positive in our study. The apoptotic index was low in
general compared to other types of tumors, but it was elevated in cases with a strong
RAR positivity. In all cases of PNET, however, a relatively high apoptotic index as well
as strong RAR positivity was found.
To the best of our knowledge, immunohistochemical demonstration of RAR has not
been reported. Our findings show that the RAR-B Ab can be used successfully also for
immunohistochemical purposes. The results suggest that spontaneous apoptotic activity
in NB may be caused by endogenous retinoic acid and mediated by RAR.
33
Tumors in Thyroid glands
Nodules in the thyroid have always commanded a great deal of attention because of
their fear of being cancerous. The female-to-male ratio is about 3 to 4:1. Most of these
solitary masses prove to be dominant nodules within a multinodular goiter, cysts, or
asymmetric enlargements of various non- neoplastic conditions, such as Hashimoto`s
thyroiditis. When such nodules prove to be neoplastic, in well over 90% of instances,
they are adenomas. Virtually all adenomas of the thyroid are present as solitary, discrete
masses. With rare exception, they all are derived from follicular epithelium and so
might all be called follicular adenomas (134). Microscopically, a variety of patterns can
be identified that recapitulate stages in the embryogenesis of the normal thyroid, and so
they have been divided into fetal, embryonal, simple, and colloid subtypes or, more
simply, into microfollicular and macrofollicular patterns. There is little virtue in these
classifications because mixed patterns are common, and ultimately all have the same
clinical and biologic significance. Numerous studies have made it clear that adenomas
are not forerunners of cancer except in the exceptional instance.
Malignant Tumors in Thyroid glands
Thyroid carcinoma is two to three times more common in middle aged women than in
men. Before puberty, there is no sex difference, and the female preponderance
disappears in postmenopausal life. Possibly relevant is the fact that most well-
differentiated thyroid carcinomas have estrogen receptors. The morphologic variants of
thyroid carcinoma with their approximate frequencies are as follows:
? Papillary carcinoma 75-85%
? Follicular carcinoma 10-20%
? Medullary thyroid carcinoma 5%
? Anaplastic carcinoma (rare)
Papillary Carcinoma, is the most common type of thyroid carcinoma, comprising 45%
to 70% of all cases (123,135). It is a slow-growing tumor that usually appears clinically
as a solitary thyroid nodule. The primary tumor is in most cases limited to the gland, but
34
especially in older people it may invade the surrounding structures, such as muscles, the
esophagus, or the larynx. The prognosis of papillary carcinoma is relatively good, with a
10-year survival rate in the order of 80% to 95% (135,136,137). Histologically papillary
carcinoma is composed of papillae, usually accompanied by follicules and sometimes
by solid sheets of cells. The papillae are often long and slender and have a fibrovascular
core that is usually thin but may be thick and hyalinized. The nuclei of papillary
carcinoma often overlap and have many typical features including a pale outlook, large
size compared with normal follicular cells, irregular outline with deep grooves and
cytoplasmic pseudoinclusions, rare mitosis, and an inconspicuous nucleolus
(138,139,140).
Follicular carcinoma comprises about one fourth of all thyroid carcinomas. It is more
common in females than in males and is a disease of middle and old age. Distant
metastases are common, occurring in as many as 25% of patients at diagnosis (141).
Their most common sites are the bones and lungs. Histologically the tumor is often
encapsulated. The capsule usually contains large, thin-walled blood vessels. Vascular
invasion -often to these vessels- is common, a finding that explains the frequent
occurrence of distant bloodborne metastases in follicular carcinoma. Follicular
carcinoma shows follicular differentiation but lacks the diagnostic features of papillary
carcinoma (142). Like follicular adenoma, follicular carcinoma is composed of
follicular, trabecular, or solid structure (143).The degree of follicular differentiation
varies, but usually the follicles are smaller and less well differentiated than the follicles
in papillary carcinoma. Follicular carcinoma is divided into two subtypes: minimally
invasive (also called encapsulated) and widely invasive (142). In the first subtype the
tumor is encapsulated, and only minimal vascular or capsular invasion is seen. In the
second subtype the tumor is either nonencapsulated or encapsulated with considerable
invasion. These two subtypes should be distinguished in prognosis (144) because the
10-year survival rate for the first type is 80% to 95% and for the second type 30% to
45% (135,136,145).
Medullary Carcinoma basically differs from other thyroid carcinomas in showing
evidence of C-cell differentiation. Like normal C-cells, the tumor cells produce and
secrete calcitonin. The tumor may also produce prostaglandins histaminase (146), and
express carcinoembryonic antigen (CEA) (147), and occasionally excretes 5-
35
hydroxytryptamine (5_HT) or adrenocorticotropic hormone (ACTH). Although most
medullary carcinomas occur sporadically, about 10% have a genetic background (148).
The sporadic carcinoma is generally unilateral but the familial tumor is usually bilateral
and multicentric and often accompanied by C-cell hyperplasia, which precedes the
development of medullary carcinoma (149,150). Also, the associated
pheochromocytoma is usually bilateral (151). Medullary carcinoma is rather rare,
comprising about 5% to 10% of all thyroid carcinomas. It is as common in men as in
women and is usually detected at middle age. The most frequent finding is a solitary
thyroid nodule, but sometimes enlarged cervical lymph nodes may be the first symptom.
Regional lymph node metastases are common; they have been detected in about half the
patients at the time of surgery (152). There are often irregular calcifications in the
stroma that may resemble psammoma bodies but do not show regular laminations. It is
not always possible to find amyloid in medullary carcinoma. In such cases the diagnosis
can be confirmed by use of silver stains for argyrophil cells (153) or
immunohistochemical methods based on the calcitonin content of the tumor cells (154).
Mixed medullary-follicular carcinoma (156,157) denotes a very rare tumor with
morphologic features of medullary carcinoma with immunoreactive calcitonin,
accompanied with morphologic features of follicular carcinoma with immunoreactive
thyroglobin.
Anaplastic carcinoma: Fortunately fewer than 5% of thyroid carcinomas fall into this
category. They tend to occur in elderly individuals, particularly in endemic goitrous
regions. There are basically three histologic paterns: 1) spindle cell carcinomas, 2) giant
cell leions, and 3) rarest of all the small cell carcinoma (155). All grow quite rapidly and
are usually large masses by the time they come to clinical attention.
II.Apotosis in Thyroid glands
Our studies on apoptosis in thyroid gland tumors
This study has been carried out by the co-operation of the:
National Institute of Oncology, Budapest, Hungary with the full support of Dr. Ilona
Peter, whom without her assistance the work would have been impossible. And the
36
Institute of Parthology, University of Debrecen, Debrecen, Hungary, where the help of
Prof. Dr. Szabolcs Gomba is deeply appreciated.
Introduction
Thyroid nodules are found in as many as 1% to 10% of the population, but malignant
tumors of the thyroid account for only about 1% of all cancer and 0.4% of cancer
related death (156). Females are affected three times as often as males (106). The aim of
this study was to measure spontaneous apoptosis and the expression of some oncogenes
which influence this process: Bcl-2 by securing cell survival (5), mutant P53 by
suppressing and bax by promoting cell death (22). With the knowledge that Bcl-2
inhibits apoptosis in a number of different cells and bax is antagonizing the protective
effect of Bcl-2 it becomes clear that prolonged survival of cells overexpressing Bcl-2 is
a factor in predisposition to malignancy (5). Nevertheless, investigation of these
oncogenes in benign tumors-adenomas could indicate a chance of malignancy in such
tumors.
Materials & Methods
Sixteen cases of thyroid carcinoma and nine cases of adenoma were investigated in a
retrospective study. Basic data (age, sex, tumor type) were registered (table 3.).
No Age
(year) Sex
Tumor type Apoptosis %
P53 %
Bax
%
Bcl-2 %
1. 24 F F. adenoma 0 2.6 12.7 Neg. 2. 35 F F. adenoma 0 4.9 1.9 Neg. 3. 44 F F. adenoma 0 0.9 0 Neg. 4. 39 F F. adenoma 0 0 11.9 Neg. 5. 45 F F. adenoma 0 33.1 43.5 Neg. 6. 55 F F. adenoma 0 5.1 0 59 7. 20 F F. adenoma 0 0.6 0 19 8. 50 F F. adenoma 0 0.3 9.2 47 9. 51 F F. adenoma 0 3.5 40.5 Neg. Subtotal 40 F. adenoma 0 5.7 13.3 41.7 Table 3.a.1. Percentage of P53, bax, bcl2 and apoptosis in thyroid adenoma
37
No Age
(year) Sex
Tumor type Apoptosis %
P53 %
Bax
%
Bcl-2 %
10. 64 F F.ad. carcinoma 0 94 12.3 Neg. 11. 50 F F.ad. carcinoma 0 22.8 4.8 2 12. 68 M F.ad. carcinoma 0 46.7 0 Neg. 13. 71 F F.ad. carcinoma 0 53.1 1.6 Neg. 14. 67 F F.ad. carcinoma 0 100 0 3 15. 55 M F.ad. carcinoma 0 81.7 1.1 Neg. 16. 17 M F.ad. carcinoma 0 100 4.2 Neg. Subtotal 56 F.ad. carcinoma 0 71.1 3.4 2.5
No Age
(year) Sex
Tumor type Apoptosis %
P53 %
Bax
%
Bcl-2 %
17. 64 F P.ad. carcinoma 0 0 3.4 Neg. 18. 44 F P.ad. carcinoma 0 100 5 Neg. 19. 43 M P.ad. carcinoma 0 0.6 4.6 Neg. 20. 17 M P.ad. carcinoma 0 0.6 0 Neg. 21. 11 F P.ad. carcinoma 0 26 0 2 22. 23 M P.ad. carcinoma 0 0 13.1 Neg. 23. 65 F P.ad. carcinoma 1% 53.1 3.2 Neg. 24. 42 M P.ad. carcinoma 0 96.1 0 Neg. 25. 72 F P.ad. carcinoma 0 0.5 1.2 4 Subtotal 42 P.ad. carcinoma 0 39.6 3.4 3
Average Age (year)
Tumor Type Apopto-sis %
P53 Box Bcl - 2
40 ? 12 F. adenoma 0 5.7 ? 10,4 13.3 ? 10,0 41.7 ? 20,5
56 ? 18 F.ad.carcinoma 0 71.1 ? 30,4 3.4 ? 4,6 2.5 ? 0,7
42 ? 22 P.ad.carcinoma 0 39.6 ? 44,3 3.4 ? 4,1 3.0 ? 1,4
The tumor tissues were obtained by surgical excision, and after formalin fixation
and paraffin embedding 8 ? m thin sections were cut. All sections were examined with
HE staining. The criteria described by Wyllie (3) were used to recognize apoptotic cells.
ApopDetec (simply sensitive In situ Detection System Kit, DAKO, Denmark) was also
used to show apoptosis. Sections were treated with proteinase K to make the tissue
Table 3.a.2. Percentage of P53, bax, bcl2 and apoptosis in thyroid follicular adenocarcinoma
Table 3.a.3. Percentage of P53, bax, bcl2 and apoptosis in thyroid papillari adenocarcinoma
Table 3.b. Average of P53, bax, bcl2 and apoptosis in thyroid tumors
38
permeable to the reagents of the labeling and detection procedure. The apoptosis index
in the tumor tissue was determined by examining 2000 tumor cells. The sections were
examined for mutant p53, bax and bcl-2 by immunoperoxidase reaction .
All antibodies to p53, bax and bcl-2 were the products of DAKO (Denmark) and were
used in dilution 1:100 overnight at 37 ? C. The sections were treated with proteinase K
and, to inactivate endogenous peroxidase, incubated in methanol and H2O2. The
secondary antimouse IgG (product of DAKO) was used in dilution 1:100 the next day.
DAB (diaminobenzidine) served as a chromogen and methyl green as counterstain. p53,
bcl-2 and bax positivity were determined by examining 2000 cells.
Results
In adenomas p53 positivity was 5.7% (fig. 6.), bax positivity was 13.3% and bcl-2
positivity was 41.7%. As for the follicular adenocarcinomas p53 showed 71.1%
positivity (fig. 7.), bax was positive in 3.4% and (Fig. 6.)
0
10
20
30
40
50
60
70
80
bcl2 bax p53
FollicularadenomaFollicularcarcinomaPapillarycarcinoma
Fig. 6. Percentage of bcl2, bax and P53 in thyroid tumors
?20,5
?0,7 ?1,4
?10
?4,6 ?4,1
?10,4
?30,4
?44,3
39
bcl-2 was positive in 2.5%. In
papillary adenocarcinomas
(Fig.7,8,9,) the value for p53
was 39.6%, for bax was 3.4%
positivity and for bcl-2 was
3%. Apoptosis was almost
absent in all cases.
Discussion
Data from this study on
mutant p53, bcl-2 and bax
expression in benign and
malignant thyroid tissue
reveal considerable results.
Mutant p53 is very low in
Fig. 7. Abundant P53 positivity in cell nuclei of a follicular adenocarcinoma. Immunperoxidase (x200)
Fig. 9. P53 positivity in papillary adenocarcinoma
Fig. 8. P53 positivity in follicular adenocarcinoma. Immunoperoxidase (x200)
40
adenomas compared to its
expression in papillary and follicular carcinomas. Bcl-2 expression is high in adenomas
and low in carcinomas according to our data. Bax expression on the other hand, is
relatively high in adenomas but still lower than bcl-2 and it is low in carcinomas.
In agreement with our results, bcl-2 and bax had been reported to be co-expressed in
aggressive thyroid carcinomas (159). As for the positivity of bax expression in papillary
carcinomas Branet et al (5) obtained similar results as described here by us. It has been
supposed that when bcl-2 is present in excess, cells are protected against apoptosis
(159,160,161). However, when bax is in excess, cells are susceptible to apotosis. As
apoptosis is concerned, only very few apoptotic tumor cells could be detected in our
study in any of the benign or malignant tumors. This may be relevant to the high
expression of bcl-2. On the other hand, high expression of bcl-2 in adenomas suggests
the susceptibility to transformation to malignancy. From a practical point of view,
determination of p53, bcl-2 and bax ratio in thyroid tumors may contribute to the
differentiation between adenomas and especially follicular carcinoma (7).
41
Fig. 11. P53 positivity in follicular adenocarcinoma. P53 immunperoxidase (x600)
Fig. 12. Bcl-2 positivity in follicular carcinoma. Bcl2 immunperoxidase (x300)
42
With the request of WHO-IARC (International Agency for Research on Cancer), after
the publication of our paper on thyroid tumours (Farid P, Gomba SZ, Peter I, Szende B.
Bcl-2, P53, and Bax in thyroid tumours and their relation to apoptosis. Neoplasm 2001;
48(4): 299-301 the following text was sent, along with a chart (fig.6) and a photo
(fig.11) and was accepted for publication in the WHO book on Pathology and genetics
of tumours of Endocrine organ.
Thyroid follicular carcinoma, Somatic genetic, expression
profiles/proteomics
Follicular carcinoma is a much more aggressive lesion than papillary carcinoma. This
cancer occurs more often in females with a peak incidence in the fifth and sixth decades.
Mortality rates are 70% at 5 years.
There is no sign of increased apoptosis, or programmed cell death, as defined by
morphological change in non-pathologic cell loss and relevant to a wide spectrum of
biology; in follicular carcinoma, however as shown by immunohistochemical method
(presumably mutant) p53, bax, and bcl-2 were expressed.
When bcl-2 is present in excess, cells are protected against apoptosis (4,5,7) since it
secures cell survival (2). Mutant p53 suppresses, while bax promotes cell death (6,1).
When Bax is in excess, cells are susceptible to apoptosis. BCL-2 and Bax had been
reported to be co-expressed in aggressive thyroid carcinoma (4).
From a practical point of view the determination of P53, BCL-2 and Bax ratio in thyroid
tumors, contributes to the differentiation between adenomas and especially follicular
carcinomas (3). In adenomas the rate of P53 positive cells was 5.7%, while it was
13.3% for Bax and 41.7% for Bcl-2 in the tumor cell. The identification of the above
mentioned oncogenes, and many more, is facilitated by proteomics, that studies the
proteins in general and in particular their changes resulting from various disorders or the
effect of external factors (8).
43
References
1. Basolo F, Pollina L, Fontanini G, Fiore L, Pacici F, Baladanzi A. Apoptosis
and proliferation in thyroid carcinoma: correlation with Bcl-2 and P53
protein expression. Brit J Cancer 1997; 74: 537-541.
2. Lumachi F, Basso S. Apoptosis, life through planned cellular death
regulating mechanisms, control systems, and relations with thyroid disease.
Thyroid 2002; 12 (1): 27-34.
3. Farid P, Gomba Sz, Peter I, Szende B. Bcl-2, P53 and Bax in thyroid tumors
and their relation to apoptosis. Neoplasm 2001; 48 (4): 229-301.
4. Manetto V, Lorenzini R, Cordon-Cardo C, Krajewski S, Rosai J, Reed CJ,
Eusebi V. Bcl-2 and Bax expression in thyroid tumors. Virch Arch 1997;
430: 125-130.
5. Muller-Hocker J. Immunoreactivity of p53, Ki67, and Bcl-2 in oncocytic
adenomas and carcinomas of the thyroid gland. Hum Pathol 1999; 30: 926-
933.
6. Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with
a conserved homolog, Bax that accelerates programmed cell death. Cell
1993, 74: 609-619.
7. Pierotti MA. Bcl-2 protein expression in carcinoma originating from the
follicular epithelium of the thyroid gland. J Pathol 1994; 172: 337-42.
8. Fountoulakis M. Proteomics: Current technologies and applications in
neurological disorders and toxicology. Amino Acid 2001; 21(4): 363-81.
9. Czyz W, Kuzdak K, Pasieka Z, Timler D, Brzezinski J. P53, MDM2, Bcl-2
staining in follicular neoplasms of the thyroid gland. Folia Histochem
Cytobiol 2001.
44
III. Tumors of the Parathyroid glands Normal parathyroid glands: In adults, most of the parathyroid glands are composed of
chief cells, but these give rise to several transitional forms as will be evident
subsequently. The chief contain lipofuscin pigment and secretory granules of
parathyroid hormones (PTH). Sometimes these cells have a “water clear” appearance
owing to lake of glycogen. Oxyphil cell and transitional oxyphils are found throughout
the normal parathyroid either singly or in small clusters.
The metabolic function of PTH in supporting the serum calcium level can be
summarized as follows, as a short review for better understanding the dysfunctions and
hyperplasia of the parathyroid glands (158):
1. PTH mobilizes calcium from bone.
2. It increases renal tubular reabsorption of calcium, thereby conserving it.
3. It promote renal production of 1,25-(OH)2D3, active in intestinal
absorption of calcium.
4. It lowers the serum phosphate level by enhancing phosphaturia.
Adenoma
Almost always solitary adenomas average 0.5 to 5.0 gm in weight but maybe much
larger. Although two may be present concomitantly in the same or different glands,
nodular hyperplasia must be ruled out when more than one adenoma is present. As a
rule, normal parathyroid parenchyma contains evenly distributed cytoplasmic fat
droplets, whereas adenomas do not contain such a fat, and hyperplastic parenchyma has
reduced or absent fat (162). The adenomas are well-encapsulated, soft, tan to red
lesions. Most are composed predominantly of chief cells, but often they contain foci of
oxyphil and transitional cells usually present as islands within a background of the chief
cells. Although oxyphil cells are traditionally thought to be non-functional, rarely a
hyperfunctioning adenoma is composed of these cells. In larger lesions, there may be
areas of infarct necrosis or hemorrhage.
45
Primary hyperplasia
Primary hyperplasia may occur sporadically or in the MEN syndromes I and II a. The
hyperplasia may be either diffuse or nodular and most often is composed predominantly
of chief cells but sometimes water clear cells and, in almost all instances, islands or
nodules of oxyphils. Poorly developed, delicate, fibrous strands may envelope the
nodules. The cells in the diffuse hyperplasia or within the nodules are arranged in a
wide variety of patterns: solid sheets, nests, trabeculae, or sometimes follicular
structures. One of the more distinctive features of primary hyperplasia is the dispersal of
occasional fat cells throughout the hyperplastic areas, but overall the total amount of fat
is reduced (163,164).
Secondary Hyperplasia
The syndrome most often appears in patients with renal failure ( uremic
hyperparathyroidism) but also with marked vitamin D deficiency or osteomalacia. The
origin of hyperparathyroidism in renal failure is attributed to phosphate retention and
hypocalcemia leading to compensatory parathyroid hyperfunction. In addtion, the renal
disease may contribute to the hypocalcemia by 1) a reduction in the synthesis of 1,25-
(OH)2D3 with impaired intestinal absorption of calcium and 2) some poorly understood
state of skeletal resistance to the calcemic action of vitamin D and PTH. Uremic
hyperparathyroidism may be observed in the absence of hypocalcemia. Similarly,
vitamin D deficiency may directly stimulate PTH secretion. Whatever the precise
mechanism, it is clear that patients with renal failure and rarely the other conditions
mentioned develop secondary parathyroid hyperfunction with hyperplasia principally of
the chief cells. In many instances, the glands revert to normal if the basic clinical
derangement is brought under control by, for example renal transplantation. With long
standing secondary hyperplasia, however, reversion to normal may not occur,
contributing to the possibility that secondary hyperplasia may in time convert into
autonomous adenoma formation, sometimes referred to as tertiary hyperparathyroidism
(165,166).
46
Parathyroid carcinoma
Fewer than 5% of cases of PHPT are caused by carcinomas. These tumors present as
gray-white, irregular masses that sometimes exceed 10 gm in weight and enlarge
usually one parathyroid gland. Many are small, however, and readily mistaken for an
adenoma. Histologically, the neoplastic cell may be of variable size and shape but are
usually remarkably uniform and not too dissimilar from normal parathyroid cells. There
is general agreement that a diagnosis of carcinoma based on cytologic detail is
unreliable, and local invasion and metastasis constitute the only reliable criteria of
malignancy (167).
III. Apoptosis in parathyroid tumors Our studies have been done with the co-operation of the Department of Surgery and
Transplantation, Semmelweis University, Budapest-Hungary, our gratitude goes to Prof.
Dr. Ferenc Perner and dr. Gyula Végso.
Hyperplastic and adenomatous lesions of the parathyroid glands are not uncommon, but
malignant tumors of this gland are extremely rare (168). Hyperplasias may be primary
lesions but in most cases parathyroid hyperplasia is the consequence of chronic renal
failure. Adenomas may appear as a subject of both primary and secondary
hyperparathyroidism (HP), but the majority of these tumours appear without anamnestic
data on renal disease (169). Of course, renal damage developes regularly as an effect of
elevated serum Ca level in HP.
Differential diagnostic difficulties are well known concerning hyperplasia, adenoma and
carcinoma of the parathyroid gland. The most important sources refer to morphological
citeria and biological behaviour of the tumours (170). The significance of DNA diploidy
in indicating the benign or malignanat character of parathyroid tumours was studied by
Bocsi et al (171). They found that DNA index had no value in deciding the benign or
malignant character of a given sample.
A few but important data are available on p53, bcl-2 expression in parathyroid
hyperplasia, adenoma and carcinoma. bcl-2 and p53 expression was found both in
hyperplasias and adenomas but carcinomas failed to express bcl-2 (172,173). The role
of other gene products in the differential diagnosis of parathyroid neoplasia like P27,
cyclin D1, and Ki67 was also emphasized (174,175,176).
47
We investigated the mitotic and apoptotic activity and also the expression of P53, Bcl-2
and Bax in hyperplasia and benign as well as malignanat tumours of the parathyroid
glands. The aim of this study was to look for differences in this respect between the
above-mentioned proliferative alterations and further to find out whether the prevalence
of enhancing or inhibitory factors of apoptotic activity may explain the rarity of
parathyroid carcinomas.
Materials & Methods Clinical and laboratory parameters: The parathyroid lesions presented here were clinically diagnosed and surgically
removed at the Clinical Department of Transplantation and Surgery of the Semmelweis
University, Budapest Hungary. Altogether 107 patients were operated on because of
hyperparathyroidism (HP), 55 proved to be primary (PH), 52 suffered from secondary
hyperparathyroidism (SH). Clinical symptoms and laboratory parameters (serum Ca and
P, creatinine and parathormone level as well as radiological examination) leading to
diagnosis of HP were registered together with the period in time between the onset of
clinical symptoms or the start of hemodialysis or the date of renal grafting and the
diagnosis. Localisation and number of enlarged and removed parathyroid glands;
postoperative serum Ca and parathormone levels were also registered.
Histological studies:
All removed parathyroid glands were fixed in buffered neutral formalin and embedded
into paraffin. 8µm thin sections were cut and stained with hematoxylin and eosin.
Immunoperoxidase reactions to detect P53, Bcl-2 and Bax as well as TUNEL reaction
to show apoptosis were performed. Anti P53, anti-Bcl-2 and anti-Bax antibodies were
the product of DAKO (Glostrup, Denmark). The antibodies were diluted 1:100 and
applied overnight at 37ºC. Sections were treated with proteinase K, incubated in
methanol and H2O2. The secondary anti-mouse IgG (DAKO, Glostrup, Denmark) was
used in dilution 1:100 the next day. Diaminobenzidine (DAB) served as chromogen and
methyl green as counterstain. The TUNEL reaction was performed using Apop Detec
(Simply sensitive In Situ Detection System Kit, DAKO, Glostrup, Denmark) which
detects DNA fragmentation along nucleosomes and stain the nuclei of apoptotic cells.
48
Cellular constitution of the parathyroid lesions and mitotic index (cosidering 1000-cells)
were determined using H&E stained sections. The criteria described by Roth (168) were
applied to differentiate between hyperplasia, adenoma and carcinoma. Apoptotic index
was established after TUNEL reaction considering 1000 cells. Both mitotic and
apoptotic indices were expressed in percent. bcl-2 and bax, as well as P53 expression
was studied considering chief cells, oxyphil cells, and clear cells. Positivity was stated if
50 percent or more of a cell type showed cytoplasmic or nuclear reaction with DAB.
Results
The age and sex distribution and the per cent of adenomas, hyperplasias and carcinomas
are shown in fig. 13.a and 13.b. The overwhelming majority of patients with primary
lesions was female. SH occurred in females and males in nearly equal number. The age
of patiens suffering from PH was in average higher than that of patients with secondary
hyperparathyroidism.
0
10
20
30
40
50
60
70
80
a hy cc
adenoma
hyperplasia
carcinoma
0102030405060708090
a hy m
adenoma
hyperplasia
mixed (adenoma andhyperplasia)
Total: 55 6 males 49 females Age: 23-70 32-76 (59,2) (60)
Total: 52 25 males 27 females Age: 14-71 20-71 (49,5) (43,3)
Fig. 13.a. Primary hyperparathyroidism age, sex distribution and percent of adenomas, hyperplasias and carcinomas
Fig. 13.b. Secondary hyperparathyroidism age, sex distribution and percent of adenomas, hyperplasias and mixed lesions
49
The majority of primary lesions were adenomas and only three carcinomas were found.
In case of secondary lesions hyperplasia was the most common finding.
The clinical symptoms and events leading to the diagnosis of HP are shown in table 4.a.
PH resulted in nephrolithiasis and in osteoporosis as well as other bone and joint
alterations in a high number of cases. SH was predictable by the fact of pre-existing
chronic renal failure treated by dialysis or renal transplantation. We have to mention
that no MEN cases and no vitamin D deficiency or marked osteomalcia cases were
found in our material. The most characteristic laboratory findings are listed in table 4.b.
PH SH
Symptoms and
events
No of cases Symptoms and events No of cases
Nephrolithiasis
Osteoporosis
Bone fracture
Bone-joint pain
Elevated se Ca, P
gastrointestinal
23
25
3
13
9
5
Osteoporosis
Bone-joint pain
Bone fracture
Nephrolithiasis
Soft tissue calcification
Gastrointestinal
Renal graft
Dialysis
Renal graft plus renal
failure
Renal failure
14
16
5
1
3
2
26
19
6
1
Table 4.a. Clinical symptoms and events leading to the diagnosis of HP
50
PH SH
Laboratory
findings
Pre-operative Post-
operative
Pre-operative Post-operative
Se Ca (mmol/l)
Se P (mmol/l)
Se PTH (pg/ml)
Se creatinine
(dialysis)
(? mol/l)
Se creatinine
(renal graft)
(? mol/l)
3(2,73-3,5)
0,83 (0,59-1,24)
247,9 (68,6-1301)
2,23 (1,4-2,62)
20,3 (1,86-49,2)
2,74 (2,2-3,21)
1,71 (0,63-3,0)
821,9(77,3-2510)
769 (180-1194)
183 (95-338)
2,14 (1,44-2,7)
50,6 (1,2-251)
High serum Ca and P levels as well as PTH level were registered in both PH and SH
cases, higher values being charateristic to SH. Preoperative serum Ca and PTH levels
reflected high PTH activity. Definitive preoperative diagnosis was secured by
ultrasound and/or CT imaging as well as by isotope scanning (Table 5.).
PH SH
Ultrasound 35 34
CT 39 41
Isotope 54 48
One of the above 7 4
Two of the above 24 24
Three of the above 24 24
Table 4.b..Characteristic laboratory findings in HP patients
Table 5. Number of cases diagnosed and localized by ultrasound, CT and isotope techniques
51
In 34 cases of PH postoperative Ca administration became necessary. Subcutaneous
autotransplation of parathyroid tissue was performed postoperatively in 13 cases of SH.
Serum creatinine level was high in nearly all SH patients with chronic renal disease.
The time gap between the onset of symptoms in PH was relatively long (5.6 years)
calling the attention to the difficulties of diagnosis of HP. The period between the start
of diagnosis on the renal grafting was in average 4,2 (Table 6.) and 9 years,
respectively.
The number of the removed parathyroids is indicated in table 7. Primary lesions were
solitary in the majority of cases and were localized in the inferior right and left
parathyroids.
PH 5-6 (0,5-30)
SH (start of dialysis) 4-2 (0,25-13)
SH (renal grafting) 9 (1,5-28)
PH SH
No of enlarged
parathyroids
No of cases No of cases
1
2
3
4
48
2
4
1
5
8
16
23
Postoperative complications such as bleeding, infection,recurrent laryngeal nerve
damage occurred in altogether 6 cases. Regarding HP all operations for PH and all but
two operations for SH proved to be successful.
Table 6.Time gap between the onset of clinical signs and events and the diagnosis of HP (years)
Table 7.Number of the parathyroid lesions
52
Histological examination of the removed parathyroids showed the structural and cellular
features of hyperplasia, adenoma or carcinoma (see Fig 13 a, b).
The typical histological appearance of an adenoma and the immunostaining for
parathormone are shown in figure 14. a. and 14. b.
Fig. 14. a. Parathyroid adenoma HE (x300)
Fig. 14. b. Parathormone adenoma. Parathormone immunperoxidase (x300)
53
The cellular constitution of adenomas and hyperplasia is shown in fig. 15. Chief and
oxyphil cells were both present in the lesions and clear cell component was also found
in hyperplasia. No difference could be detected between the primary and secondary
lesions in this respect.
Apoptotic index was equally low in adenomas and hyperplasias and so was the mitotic
ratio. The carcinomas showed slight elevation in mitotic and apoptotic activity (fig. 16).
0102030405060708090
adenoma hyperplasia
oxyphilchiefclear
0
1
2
3
4
hyperplasia adenoma carcinoma
apomito
±0,1
±0,02
±0,15
±0,02
±0,2
±0,3
Fig. 16. Apoptotic and mitotic index.
Fig. 15. Distribution of cells types in hyperplasia and adenoma.
±7 ±6
±4
±7
±3
54
The ratio of P53, Bcl-2 and Bax expression was studied considering chief cells, oxyphil
cells and clear cells. Hyperplasias showed p53 positivity in the oxyphil cells in 10% of
the cases, chief cells were positive in 5 percent. The positivity was nuclear in general,
but in some cases cytoplasmic positivity was seen. The ratio of bcl-2 positivity was 60
percent in the oxyphil, 20 percent in the chief and 10 percent in the clear cells, on
average.
The bax expression was ten percent higher in all types of cells, compared to bcl-2
expression (fig. 17.a.,17.b.).
Fig. 17.a.
Fig. 17.b.
55
Figure 18.a. shows Bcl-2 positive cells in hyperplasia. bax positive cells in
hyperplasia are shown in figure 18. b. Co-expression of bcl2 and bax, as well as p53
was registered in most of the positive cases. Adenomas were positive for p53 in 15
percent of the cases, considering oxyphil cells and in 5 percent of the cases
considering chief cells. Positivity appeared in the nucleus in the majority of cases
but in few cases cytoplasmic positivity was observed (Fig. 19.a, 19. b).
Fig. 18.a.parathyroid hyperplasia, Bcl2, immunoperoxidase (x300)
Fig. 18.b. Bax positivity in hyperplasia. Bax immunperoxidase (x300)
Fig. 19.a. P53 nuclear positivity in parathydoid adenoma. P53 immunperoxidase (x300)
56
The expression of bcl-2 and bax in the oxyphil cells of adenomas was equally high and
a relatively high expression of bcl-2 and bax was present in the chief cells. Figure 20.a.
and 20. b. shows bcl-2 and bax positive cells of adenomas, respectively. Co-expression
of the three genes was characteristic to adenomas as well.
Fig. 20.a. Bcl2 positivity in parathyroid adenoma. Bcl2 immunperoxidase (x300)
Fig. 20.b. Bax positivity in parathyroid adenoma. Bax immunperoxidase (x600)
57
The few cases of carcinomas showed nuclear p53 positivity (Fig. 21.a.) and the
cytoplasm was negative for bcl-2 and bax (Fig. 21.b.).
Fig. 21.a. P53 nuclear positivity in parathyroid cancer. P53 immunperoxidase (x 300)
Fig. 21. b. Bcl2 negativity in parathyroid cancer. Bcl2 immunperoxydase (x600)
58
Discussion The results of our study on a relatively large number of parathyroid hyperplasias and
adenomas clearly indicate that mitotic or apoptotic activity can not differentiate between
these two pathological entities. Both mitotic and apoptotic indices were equally low in
hyperplasias and adenomas. In accordance with the study of Ricci et al (172) p53 and
bcl-2 expression was found in hyperplasias as well as adenomas. The percent of cases
considered as positive for p53 or bcl-2 was slightly higher in adenomas, but differential
diagnosis can not be based on p53 or bcl-2 immunostaining. The same may be applied
to bax expression. According to our knowledge expresion of bax has not been studied
yet in proliferative parathyroid lesions. The most interesting finding in our study was
the co-expression of bcl-2 and bax in most hyperplasias and adenomas. Oxyphil cells
and chief cells were studied separately regarding p53, bcl-2 and bax expression.
Oxyphil cells showed positivity for these gene products in a significantly higher
proportion of cases than chief cells or modified (clear) chief cells. This fact may be due
to the abundance of mitochondria in oxyphil cells. The appearance of p53 positivity in
the cytoplasm of both hyperplastic and adenomatous lesions may point to the increased
activity of inhibitor of P53 amino acid terminal nuclear signal (177) which means an
increased defense against transport of mutant P53 protein into the nucleous. The
antibody used by us shows predominantly mutant P53 by immunostaining. Nuclear
positivity of some of the adenomas may be a sign of the tendency towards malignant
transformation.
The very low number of carcinomas does not allow us to draw definite conc lusions
comparing these tumours with adenomas. In accordance with the litrature (178,179)
mitotic as well as apoptotic activity were slightly elevated in carcinomas. Nuclear p53
positivity and the abscence of bcl-2 as well as bax expression in carcinomas are in
accordance with the findings of Stojadinovic et al (173). Co-expression of p53, bcl-2
and Bax may be a factor responsible for the low mitotic and apoptotic activities well as
for the very low rate of malignomas in the parathyroid glands.
Our clinical findings may contribute to the differential diagnosis of hyperplasia and
adenoma of the parathyroid glands. Single lesions are more likely adenomas than
multiple ones. Adenomas occurred overwhelmingly in females. The gender distribution
in case of hyperplasia was practically even. The average age of patients with PH was
close to 60, SH occurred in earlier years of life.
59
Serum Ca and PTH levels were significantly higher in hyperplasias compared to
adenomas. Chronic renal failure, dialysis or renal grafting are anamnestic data pointing
to SH and the likelihood of hyperplasia. The presence of bone and joint lesions in both
PH and SH and the very long time between the onset of these symptoms and the
surgical removal of the parathyroid lesions calls attention to the importance of
considering the possibility of HP first of all in case of osteoporosis.
60
Conclusions
In Neuroblastomas and PNET apoptotic index and RAR index varied parallel to
each other, i.e., when apoptosis was frequent, the RAR index was also elevated. As for
NBs, not all were RAR positive in our study. The apoptotic index was low in general
compared to other types of tumors but it was elevated in cases with a strong RAR
positivity. In all cases of PNET, however, a relatively high apoptotic index as well as
strong RAR positivity was found.
To the best of our knowledge, immunohistochemical demonstration of RAR has not
been reported yet. Our findings show that the RAR-ß Ab can be used successfully also
for immunohistochemical purposes. The results suggest that spontaneous apoptotic
activity in NB may be caused by endogenous retinoic acid and mediated by RAR.
From the practical point of view the treatment of patients with RA was dependent on the
result of our examination and it the case of positivity for the RAR the treatment was
carried out nt the clinic.
Having examined p53, bcl-2 and bax in different tumors of the Thyroid, a new
dimension has been observed. Mutant p53 is very low in adenomas compared to its
expression in papillary and follicular carcinomas, and mutant p53 suppresses cell
death. Bcl-2 expression is high in adenomas and low in carcinomas according to our
data, and bcl-2 secures cell survival. bax expression on the other hand, is relatively high
in adenomas (but still lower than bcl-2) and low in carcinomas, and bax promotes cell
death.
The high expression of Bcl-2 explains the low ratio of apoptotic cells in the examined
thyroid tumors, benign and malignant. The high expression of Bcl-2 in adenomas
suggests the susceptibility to tranformation to malignancy. Although few apoptotic
tumor cells could be detected in our study in any of the benign or malignant tumors this
may be relevant to the high expression of bcl-2, since bcl-2 is known to secure cell
survival.
From the practical point of view, determination of P53, Bax and Bcl-2 ratio in thyroid
tumors contributes to the differentiation between adenomas and especially follicular
carcinomas.
As to the parathyroid lesions the overwhelming majority of patients with primary
lesions were female. Secondary hyperparathyroidism occured in females and males in
61
nearly equal numbers. The age of patients suffering from PH was on average higher
than that of patient s with SH. The majority of primary lesions were adenomas and only
three carcinomas. In case of SH, hyperplasia was the most common finding.
Apoptotic index was equally low in adenomas and hyperplasia and so was the mitotic
ratio. The carcinomas showed slight elevation in mitotic and apoptotic activity.
The results of our study on a relatively large number of parathyroid hyperplasias and
adenomas clearly indicate that mitotic or apoptotic activity cannot differentiate between
these two pathological entities. Bcl-2 and bax and in some cases p53 expression was
found in hyperplasias as well as adenomas. The percent of cases considered as positive
for p53 or bcl-2 was slightly higher in adenomas, but differential diagnosis can not be
based on p53 or bcl-2 immunostaining.
The same may be applied to bax expression (according to our knowledge expresion of
bax has not been studied yet in proliferative parathyroid lesions).
The most interesting finding in our study was the co-expression of bcl-2 and bax in most
of hyperplasias and adenomas.
Spontaneous apoptosis is generally low in endocrine cells, but overall knowledge in this
field reveals that hormones produced by different endocrine cells like PTH or LH
induce apoptosis. This by itself probably can build up a protective mechanism in
endocrine cells against apoptosis. The great emphasis in our study had been given to
shed light over this subject and has called our attention to the co-expression of bax and
bcl-2 and its relation to the protective mechanism against apoptosis in endocrine tumors.
62
Abbreviations
NB, Neuroblastoma
PNET, Primary neuroectodermal tumors
PCD, Programmed Cell Death
INPC, International Neuroblastoma Pathology Committee
NTs, Neuroblastic Tumors
OPEC, Oncovin+Cisplatin+Etoposid+Cyclophosphamid
OJEC, Oncovin+Cyclophosphamid+Etoposid+Carboplatin
VAIA, Vincristin+Actinomycin/Adriamycin+Ifosfamid
VAC, Vincristin+Actinomycin/Adriamycin+Cyclophosphamid
ALL-BFM-88/NHL-BFM-95,
Prednisolon+Vincristin+Daunorubicin+Cyclophosphamid+L-
Asparaginase+Leupurin+Metotrexat.
NB-A-89, Dacarbazin+Adriamycin+Mustarnitrogen+Vincristin
Newcastle, Vepesid+Ifosfamid/Vincristin+Cisplatin/Vincristin+Ifosfamid+Adriamycin
RAR, Retinoic Acid Receptor
DAB, Diaminobenzidine
PTH, Parathormone
PH, Primary Hyperparathyroidism
SH, Secondary Hyperparathyroidism
63
Acknowledgement
It seems that the journey has ended, but as Baha’u’llah said in one of his masterpiece
the seventh valley is the valley of true poverty and absolute nothingness, he added that
these journeys have no visible ending in the world of time, but the severed wayfarer- if
invisible confirmation descend upon him and the Gurdian of the Cause assist him-may
cross these seven stages in seven step, nay rather in seven breaths.
The invisible ending is when one finding leads us to search for another, and that is the
beauty of research work. The invisible confirmation was deeply felt, and the heart-
warming fact that I indeed was not alone on this journey was a daily experience. I
would like to express all my respect, gratitute and appreciation to beloved Professor
Bela Szende without whom none of the my imagination would take the color of reality.
His patience and his love to the research work has been exemplary. I greatly appreciate
the possibilities given by Professor László Kopper the head of the department for
fullfiling my dream in the 1st Department of Pathology and Experimental Cancer
Research. My utmost thank to Professor András Jeney for his continous support. I
would like to thank my dear husband Dr.Ajang Farid, for being extremely supportive
and for loving me for who I am. My hearfelt appreciation goes to our assistant in the
laboratory Dr.Paczolay Gyozone and dear Mrs.Erzsebet Kiss who has been a great help
with the computer work. I also would like to thank Mr.Andrew Singer for proof reading
my paper with the accuracy he did.
64
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