ultraviolet-induced transformation of keratinocytes: possible involvement of long interspersed...
TRANSCRIPT
Ultraviolet-induced transformation of keratinocytes: possible involvement of
long interspersed element-1 reverse transcriptase
Gautam Banerjee1, Nishma Gupta1,2, Jyoti Tiwari3, Govindarajan Raman1
1Cell and Molecular Biology, Toxicology Section, Environmental Safety Laboratory, Hindustan Lever Research Centre, Andheri, Mumbai, India,2Department of Biotechnology, Madurai Kamraj University, Madurai, India, and 3Tea Science Laboratory, Unilever Research, Bangalore, India
The normal human keratinocyte cell line, HaCaT, was
transformed using multiple doses of ultraviolet
(UV)A1B (UVA, 150–200mJ/cm2 and UVB, 15–
20mJ/cm2 � 6). Malignant transformation was con-
firmed by upregulation of Cyclin D1 (mRNA) and
formation of colonies on soft agar. To identify the genes
involved in this transformation process, we have done
rapid amplification of polymorphic DNA using RNA
from unexposed and multiple-exposed cells. Six percent
PAGE showed several differentially regulated genes in
exposed cells compared with unexposed cells. Total 19
genes were identified, cloned and sequenced. Three of
these 19 cloned genes showed 99% homology at both
DNA and protein levels to a stretch of 540 bp (180 aa) of
long interspersed element (LINE)-1 reverse transcrip-
tase (RT) open reading frame (ORF-2). Colonies from
soft agar showed upregulation of this gene compared
with non-colonized (lawn on soft agar) cells as detected
by RT-PCR. This data implicates LINE-1 RT (ORF-2)
in UV-induced malignancy and can possibly be used as a
marker for the diagnosis of UV-induced skin cancer.
Key words: HaCaT; keratinocytes; LINE-1 reverse
transcriptase; RAPD; skin cancer; soft agar; UV.
Cell death or ‘Apoptosis’ is an essential physiolo-
gical process that occurs in metazoans for their
normal growth and development, regulation of cell
turnover and as a defense strategy against invading
pathogens. Deregulation of apoptosis leads to disrup-
tion of tissue homeostasis giving rise to a variety of
disorders including cancer, neuro-degenerative dis-
eases, autoimmune diseases and diabetes (1). Mal-
function of apoptosis leading to cancer could involve
stepwise mutations in proto-oncogenes (ras, c-myc)
(2), tumor-suppressor genes (p53) (3), anti-apoptotic
genes (Bcl-XL) (4) or overexpression of genes (telo-
merase, cytokeratins) (5–7). The defective regulation
of apoptosis may contribute to the etiology of cancer,
and the impairment of normal cell death processes has
been implicated in neo-plastic transformation as
several studies have shown (8, 9).
Ultraviolet (UV) radiation in particular UVB (290–
320 nm) from sunlight represents one of the most
important external stimuli that affects skin by
inducing immuno-suppression, cancer, premature skin
aging, inflammation and cell death (10). UV radiation
from sun has also been well established as a principal
carcinogen serving as initiator and promoter for many
skin tumors (11). It can modulate both life/death
signaling processes in either in vivo or in vitro systems.
Studies have shown that UVB irradiation causes
apoptosis, necrosis, differentiation and malignancy in
keratinocytes in a dose-dependent manner (12, 13).
HaCaT cells constitute the basis of the in vitro
transformation assay for detecting carcinogens. The
end point used in this assay is the morphological
transformation of colonies that develop 21 days after
the seeding of HaCaT cells postexposure. The
relevance of the transformed phenotype as an
indicator of neo-plastic properties has been demon-
strated by their ability to produce tumor when
injected into isolog animals (14, 15).
The study aims to identify novel genes that are
regulated during this process so that they can be used
(1) as markers or (2) to identify new pathways. We
have shown that with repeated subapoptotic doses of
UV some cells become transformed cells and survive
as colonies on soft agar, while at higher doses cells dieAbbreviations: ORF, open reading frame; RAPD, rapidamplification of polymorphic DNA; RT, reverse transcriptase.
Photodermatol Photoimmunol Photomed 2005; 21: 32–39Blackwell Munksgaard
CopyrightrBlackwellMunksgaard 2005
32
by either apoptotic or necrotic pathways. In the
present report we present data on transformation of
normal human keratinocytes, HaCaT cell line by
multiple subapoptotic doses of UV. We have also
identified and characterized a gene, which is differen-
tially regulated during this transformation process.
Materials and methodsMaterials
DMEM medium, RPMI-1640 medium, antibiotic–
antimycotic solution and TRI reagent were purchased
from Sigma, St Louis, MO, USA. Fetal calf serum was
from Hy-clone, Logan, UT, USA. Reverse transcriptase
(RT) kit, Taq DNA polymerase, was purchased from
MBI Fermentas, Hanover, MD, USA. Gel extraction
and plasmids isolation kit were purchased from Qiagen,
Valencia, CA, USA. pGEM-T easy vector was pur-
chased from Promega, Madison, WI, USA. Custom-
made synthesis of primers and sequencing of DNA was
done by Microsynth, Balgach, Switzerland. LB agar
and yeast extract were from Hi-Media, Mumbai, India.
UV exposure was done using Woton lamp at a
distance of 26 cm and the dose was monitored using
UV meter (Solar Light Inc., Philadelphia, PA, USA).
HaCaT cell line was a gift from Dr. N. Fusenig,
Germany.
Methods
Cell culture: HaCaT and A431 were maintained in
complete DMEM in the presence of 10% FCS at
37 1C and 5% CO2. They were subcultured in every
3–4 days using Trypsin EDTA.
UV exposure to HaCaT cells: Confluent layers of
HaCaT cells grown in a six-well plate were exposed to
UVA1B (UVB, 15–20mJ/cm2). These cells were
cultured in fresh medium for 1–2 days and re-exposed
using the same dose. This procedure was repeated six
times. After each exposure, cells were trypsinized and
divided into three aliquots. These aliquots were used
for RNA extraction, soft agar colony assay and the
remaining aliquot was used for the next exposure.
Control cells were subjected to similar procedures
without being UV exposed.
Soft agar colony-forming assay: Soft agar colony-
forming assay using multiple-exposed cells was done
as follows. Base agar plates were prepared containing
2.5ml of 1% agar and 2.5ml of 1 � RPMI with 15%
FCS. HaCaT cells (1 � 106 cells/90mm plate) were
suspended in 1ml of RPMI with 15% FCS and 1ml
of 0.7% agar and were plated. These plates were kept
at 37 1C for 21 days and examined for colonies.
Individual colonies as well as the lawn (non-colo-
nized) cells were picked up and RNA was isolated
from these cells for further analysis.
PCR amplification: RNA was extracted from the
exposed as well as unexposed cells using TRI reagent
as per manufacturer instruction. 1.5 mg RNA was
converted to cDNA using the RT kit from MBI
Fermentas. PCR was done using the b-actin primer to
equalize the amount of cDNA for subsequent
analysis. PCR was also performed without converting
them to cDNA to determine the effect/contamination
of genomic DNA in the extracted RNA.
PCR was conducted in 10 � PCR buffer with
25mM MgCl2 supplemented with 2mM dNTP’s,
20 pmol sense and antisense primers for b-actin or
Cyclin D1, 1 ml cDNA containing the same amount
and 1 unit of Taq DNA polymerase in a 20 ml reactionmixture. PCR profile consisted of 25 cycles (for b-actin) and 35 cycles (for other genes) of 94 1C for 60 s,
57 1C for 90 s, 72 1C for 2min and final extension at
72 1C for 10min. We have checked that the condition
we have used for (for b-actin) lies in the exponential
phase and not in the stationary phase.
PCR products were analyzed on a 2% agarose gel
and stained with ethidium bromide and visualized
under UV illumination.
The sequences of the primers are given below:
b-actin
Forward 50-AGCGGGAAATCGTGCGTG
Reverse 50-CAGGGTACATGGTGGTGCC
Cyclin D1
Forward 50-AGGAGAACAAACAGATCA-30
Reverse 50-TAGGACAGGAAGTTGTTG-3 0
Rapid amplification of polymorphic DNA (RAPD):
The cDNA that were free from contamination DNA
as judged by – RT-PCR experiments were used for all
RAPD – PCR analysis. This confirms the absence of
any artifact DNA or genomic long interspersed
element (LINE)-1 sequences. The cDNA from un-
exposed and exposed cells was diluted to 1 : 50, 1 : 75
and 1 : 100. cDNA was amplified using KD1 (50-
AAGAGCCCGT-30) primer for 35 cycles as described
below – 95 1C for 5min, 36 1C for 30 s and 72 1C for
30 s followed by 35 cycles of 95 1C for 1min, 36 1C for
30 s and 72 1C for 30 s. The amplified products were
separated on 6% SDS-PAGE and silver stain was
done. The differentially expressed bands were cut out,
extracted and purified. The purified DNA was
33
Ultraviolet-induced transformation of keratinocytes
re-amplified using the same procedure. Two percent
agarose gel was used to separate the DNA and
amplified band was excised from the gel and was
extracted using gel extraction kit (Qiagen). The DNA
was ligated in pGEM T easy vector (Promega)
overnight at 4 1C. DH5a was used as host for
transformation of ligated plasmids.
Isolation of plasmids: Plasmids were isolated from
overnight culture of transformed DH5a using the spin
mini prep kit (Qiagen). The plasmids were digested
with EcoR1 to confirm the presence of insert. Once
confirmed the inserts were sent for sequencing.
Data analysis: The sequence was analyzed using the
National Center for Biotechnology (NCBI) database
and the alignments were done using Clustlaw data-
base of the European Biochemical Institutes.
LINE-1 regulation in multiple-exposed cells: LINE-1
RT sequence specific primers were designed (forward
50-ACGGTGATTTCTGCATTTCC-3 0 and reverse
50-TTTCTTAAGCCGGTCTGAAA-30) from pub-
lished sequences and PCR was done using cDNA
from exposed cells (after the fifth exposure), un-
exposed cells as well as the cells from lawn and
colonies obtained from soft agar plates.
ResultsThe generation of malignant colonies on soft agar is a
useful method to identify malignant clones. The
transformation of normal HaCaT to malignant
phenotype by UVB was investigated by soft agar
colony assay. Unexposed cells did not show any
colony even after 6 weeks. The colonies start
appearing from the fourth exposure onward. The
numbers (not detected at unexposed cells to about
2000/90mm plate after the fifth exposures) as well as
the size of the colonies increases with increasing the
number of exposure. The unexposed cells were taken
from the same passage as that of cells after the fifth
exposure, which rules out the involvement of passage
number in colony formation. A431 cells, known
carcinoma cells, were used as positive control (with-
out any UV exposure). The result is shown in Fig. 1.
All the pictures were taken in the same magnification
(� 40). A431 is a cancer cell line hence the increased
number of colony is justified, whereas small percen-
tages of HaCaT (4–5%) are converted to malignant
phenotype. Hence the numbers of colonies are less in
HaCaT and should not be compared with that of
A431cells.
The upregulation of Cyclin D1 was seen in cells
obtained from liquid culture medium (after the fifth
exposure compared with unexposed cells). The results
a b c
d e f
Fig. 1. Soft agar colony assay using ultraviolet (UV)-exposed and unexposed HaCaT cells. (a) Unexposed cells, (b)the first exposure, (c) the second exposure, (d) the fourth exposure, (e) the fifth exposure, (f) A431 cells. Confluentlayers of HaCaT cells grown in a six-well plate were exposed to UVB (15–20mJ/cm2). These cells were cultured infresh medium for 1–2 days and re-exposed using the same dose. This procedure was repeated six times for a periodof 2 weeks. Control cells were subjected to similar procedures without being UV exposed. The colonies wereallowed to grow for 21 days post last exposure. The pictures were taken at the same magnification (� 40).
34
Banerjee et al.
are shown in Fig. 2. Upregulation was observed in the
multiple-exposed cells compared with unexposed cells
from the same passage.
To identify the regulation of genes in this transfor-
mation process we have done RAPD using different
dilutions of cDNA from the exposed (the first to fifth
exposure) and unexposed cells. Differential expression
of bands of varying sizes was seen. The bands that
were either new or upregulated (500–750 bp) were
taken out for further analysis. Total 19 bands were
identified. Bands from different exposures (fourth
and fifth) with approximate 550 bp were re-amplified
and purified from a 2% agarose gel. The DNA was
ligated in pGEM -T easy vector. The presence of insert
was confirmed by digesting the plasmids with EcoR1.
The insert was sequenced (Microsynth). Three
different sets of inserts (isolated from cells post fourth
and fifth exposures) had significant homology (95%)
among themselves in both DNA as well as in protein
sequence. The protein sequence homology of the
cloned genes is shown in Fig. 3. The description of the
LINE-1 open reading frame (ORF-2) gene is shown in
Fig. 4. Blast analysis using the NCBI database
showed homology (96%) with the OFR-2 of the
LINE-1 RT (Fig. 5). Hence three out of the 19 cloned
gene were similar to LINE-1, ORF-2.
In order to identify the presence of ORF-2 of RT in
both exposed/un-exposed cells as well as in the cells
from the soft agar colonies (single colony and lawn),
a sequence-specific primers pair was designed. The
primer pair was designed in such a way that the
forward primer situated upstream of our cloned
gene and the reverse primer were inside the cloned
gene. Hence any upregulation indicates the regulation
of entire gene and not a spliced part. The ORF-2 was
upregulated in exposed cells compared with unex-
posed cells. The same assay was done using the
colonies and lawn cells. The ORF-2 RT was also
upregulated in the colonies compared with the lawn or
non-colony cells. The data is shown in Fig. 6.
DiscussionUV radiation from sun is one of the major environ-
mental agents that affects skin. Apart from its critical
role in formation of vitamin D, it adversely affects
mammalian systems by causing sunburn, suntanning,
immunosuppression and most importantly it is
implicated in induction of non-melanoma skin cancer.
Unexposed
Cyclin D1
β-actin
Exposed
Fig. 2. Upregulation of Cyclin D1. (a) Cyclin D1 inunexposed and exposed cells, (b) b-actin in unexposedand exposed cells after the fifth exposure. Confluentlayers of HaCaT cells grown in a six-well plate wereexposed to UVB (15–20mJ/cm2). These cells werecultured in fresh medium for 1–2 days and re-exposedusing the same dose. This procedure was repeated sixtimes for a period of 2 weeks. Control cells were sub-jected to similar procedures without being UV exposed.
g16 IAKTALSKKNKAGGIMLPDFKLHYKDTVTKTAGYCYQNRHIDQZNRTEPSEIRPHIYNHL 60g17 IAKTALSKKNKAGGIMLPDFKLHYKDTVTKTAGYCYQNRHIDQZNZTEPSEIRPHIYNHL 60g5 IAKTALSKKNKAGGIMLPDFKLHYKDTVTKTAGYCYQNRHIDQZNRTEPSEIRPHIYNHL 60 ********************************************* **************
g16 IFDKPDKNKKWGNDSLFNKZCWENWLAICRKLKLDPFLTPYTKINSRWIKDIHVRPETIK 120g17 IFDKPDKNKKWGNDSLFNKZCWENWLAICRKLKLDPFLTPYTKINSRWIKDIHVRPETIK 120g5 IFDKPDKNKKWGNDSLFNKZCWENWLAICRKLKLDPFLTPYTKINSRWIKDIHVRPETIK 120 ************************************************************
g16 TLKENLGNSIQDIGMGKDSMTKTAKAMATKAKIDKWDLIKLKSLCTAKKK---KKNKKNK 177g17 TLKENLGNSIQDIGMGKDSMTKTAKAMATKAKIDKWDLIKLKSLCTAKKK---QKKQKNK 177g5 TLKENLGNSIQDIGMGKDSMTKTAKSNGNKSQNZZMGSNZTKEPLPSKKKKKKQKNQKNK 180 *************************: ..*:: . *. .:***...:*::***
g16 KZTKNYHQS 186 g17 QKTIIRVK- 185 g5 QKTIIRV-- 187 : *
Fig. 3. Protein sequence alignment of cloned genes (g-5, 16 and 17). The sequence homology was done using theclustlaw software.
35
Ultraviolet-induced transformation of keratinocytes
An effective repair system that excises the photo-
products formed primarily at sites of adjacent
pyrimidines of DNA operates in cells (3). If UV-
exposed cells fail to repair the damaged DNA, then
p53-dependent apoptosis is induced in keratinocytes,
which serves as a protective mechanism against
UTR ORF1 ORF2 UTRA(n)
3’5’
pL1
1 239 380 480 498 590 773 1130 1147 1275 aa
EN Z RT CYS - rich
RT5Z8
a
b
Fig. 4. Gene sequence of long interspersed element-1 reverse transcriptase (RT). (a) Full-length gene having openreading frame (ORF)-1 and ORF-2 with non-coding region at both 50 and 30 region and a promoter region (Pl-1),(b) full length of ORF-2 �239 amino acid long endonuclease domain (EN), octapeptide repeat region (Z), RTregion and a cysteine-rich residue. The crossed bar on top of RT region indicates the cloned gene.
ORF-2 AAGTTCATATGGAACCAAAAAAGAGCCCGCATTGCCAAGTCAATCCTAAGCCAAAAGAAC 1800 g17 ------------------------------ATTGCCAAGACAGCCCTAAGTAAAAAGAAC 30
********* ** ****** ********ORF-2 AAAGCTGGAGGCATCACACTACCTGACTTCAAACTATACTACAAGGCTACAGTAACCAAA 1860 g17 AAAGCTGGAGGCATCATGCTACCTGACTTCAAACTACATTACAAGGATACAGTAACAAAA 90
**************** ****************** * ******* ********* ***ORF-2 ACAGCATGGTACTGGTACCAAAACAGAGATATAGATCAATGGAACAGAACAGAGCCCTCA 1920 g17 ACAGCAGGGTACTGTTACCAAAACAGACATATAGACCAGTAGAACTGAACAGAGCCCTCA 150
****** ******* ************ ******* ** * **** **************ORF-2 GAAATAATGCCGCATATCTACAACTATCTGATCTTTGACAAACCTGAGAAAAACAAGCAA 1980g17 GAAATAAGGCCCCATATCTACAACCATCTGATCTTTGACAAACCTGACAAAAACAAGAAA 210
******* *** ************ ********************** ********* **ORF-2 TGGGGAAAGGATTCCCTATTTAATAAATGGTGCTGGGAAAACTGGCTAGCCATATGTAGA 2040 g17 TGGGGAAACGATTCCCTATTTAATAAATGATGCTGGGAAAACTGGCTAGCCATATGTAGA 270
******** ******************** ******************************ORF-2 AAGCTGAAACTGGATCCCTTCCTTACACCTTATACAAAAATCAATTCAAGATGGATTAAA 2100g17 AAGCTGAAACTGGATCCCTTCCTTACACCTTATACAAAAATTAATTCAAGATGGATTAAA 330
***************************************** ******************ORF-2 GATTTAAACGTTAAACCTAAAACCATAAAAACCCTAGAAGAAAACCTAGGCATTACCATT 2160 g17 GACATACACGTTAGACCTGAAACCATAAAAACCCTAAAAGAGAATCTAGGCAATTCCATT 390
** ** ****** **** ***************** **** ** ******* * *****ORF-2 CAGGACATAGGCGTGGGCAAGGACTTCATGTCCAAAACACCAAAAGCAATGGCAACAAAA 2220g17 CAGGACATAGGCATGGGCAAAGACTCCATGACTAAAACAGCAAAAGCAATGGCAACAAAA 450
************ ******* **** **** * ****** ********************ORF-2 GACAAAATTGACAAATGGGATCTAATTAAACTAAAGAGCTTCTGCACAGCAAAAGAAACT 2280 g17 GCCAAAATTGATAAATGGGATCTAATTAAACTAAAGAGCCTCTGCACAGCAAAAAAAA-- 508 *
********* *************************** ************** ***ORF-2 ACCATCAGAGTGAACAGGCAACCTACAACATGGGAGAAAATTTTCGCAACCTACTCATCT 2340g17 --- AACAAAAAAAACAAAAAA---ATAA-ACAAAAAACTATCATCAGAGTGAA------- 554
* ** * **** ** * ** * * * ** ** * *
Fig. 5. Sequence alignment of open reading frame (ORF)-2 of long interspersed element-1 reverse transcriptase andcloned gene (g-17).
36
Banerjee et al.
development of cancer (3). UV-induced DNA damage
can lead to mutations that may trigger malignancy.
UV-induced skin cancers in mice models have earlier
been analyzed. It is reported that both ras and p53
genes were mutated in these models (16). As a result
of such DNA modification, the apoptosis pathway is
inhibited and malignancy allowed developing.
Mutations in tumor-suppressor gene, p53, have
been found to be characteristic in UV-induced skin
cancer. Evidences also have shown that p53 mutations
offer selective growth advantage to initiated cells.
Cells even with a single p53 mutation have less
apoptotic cell death in comparison with neighboring
p53�/� cells, thereby they continue to survive and
multiply (17). This would mimic an in vivo condition
where UV light would exert selective pressure for
mutated (p53�/�) keratinocytes hence allowing the
cells to clonaly expand and eventually become
malignant. These cells still may undergo apoptosis
by p53-independent pathways.
In this paper we have shown that repeated exposure
at subapoptotic doses of UV (UVA, 150–200mJ/cm2
and UVB, 15–20mJ/cm2) might cause transformation
in HaCaT. It is reported that HaCaT is p53�/� but it
cannot spontaneously transform during multiple
passages (18). Tumorigenic transformation in HaCaT
has been induced by ras oncogene transfection and
at elevated temperatures (18). The transformed cells
we have generated because of multiple exposures are
not because of spontaneous transformation through
multiple passages as it is reported that up to at least
300 passages this type of transformation does not take
place (18).
Telomerase is an enzyme known to protect
immortal/malignant cells from loss of telomeric
repeats at the end of chromosomes and thereby
protects the cells from death. All malignant cells/
tissues are known to contain high levels of telomerase
while the normal cells/tissues are lacking in this
enzyme (5, 6). It is reported that unexposed HaCaT
possess some basal level of telomerase activity while
those of malignant cell lines like A431 or HeLa
possess much higher amount (19). This data is
consistent with our findings on soft agar colony-
forming assay. We have shown that unexposed cells
did not induce colony formation while multiple-
exposed cells induced colony formation on soft agar
plates. With increasing number of exposures (beyond
the fourth exposure), the number and size of the
colonies increase. These data demonstrate the trans-
formation of HaCaT from non-malignant to a
malignant phenotype by multiple UVB exposure.
The neo-plastic transformation of human epidermal
keratinocytes by ionizing radiation has also been
reported and soft agar colony assay was done to
confirm the transformation by radiation (20).
LINES are most abundant mobile elements in
mammalian genome. They are non-viral retrotran-
sposons occurring as moderately repeated sequences
in the genome. Full-length version of this gene is
approximately 7 kb and include a 5 0 non-coding
region with a promoter, ORF-1 and ORF-2 and a 3 0
non-coding region ending with an A-rich stretch.
Most common LINE constitute the L1 LINE
family, the consensus sequence of which contains
ORF-2 coding for RT. RT plays an important
role in the replication of these retro elements by
reverse transcribing their template RNA into double-
strandeded DNA that is ultimately integrated into
the host genome by the element encoded integrase
(21, 22).
Human LINE-1 retrotransposons have known
implications in origin and progression of tumors by
either causing insertional mutations or chromosomal
translocations/rearrangements (23, 24). These ele-
ments have been shown to be expressed in a variety
of adult and pediatric germ cell cancers (24, 25).
Expression of these retrotransposons has been shown
to be favored in cells of germ LINE origin and also in
tumors of epithelial origin (22). LINE-1 transposition
is also shown to cause chromosomal rearrangement
giving rise to fusion transcript encoding an aberrant
transcription factor, which is implicated in desmo-
plastic round cell tumor (23, 26). RT is thus known
to have significant biological role in the etiology
of tumors.
Distinct upregulation of LINE-1 is observed in the
set of multiple-UV-exposed cells as compared with
unexposed cells. Treatment of cells with UV is known
Unexposed ExposedLane 1 2 1 2 3 4
a b
c d
Unexposed Exposed
LINE 1, ORF 2
Lawn colony colony colony
Lawn colony colony colony
β-actin
Fig. 6. Upregulation of long interspersed element(LINE)-1 reverse transcriptase open reading frame(ORF)-2 in the multiple-UV-exposed cells. (a,c) Lane-1: unexposed cells, lane-2: exposed cells. (b,d) Lane-1:non-colonized cells (exposed) from soft agar plate,lanes 2–4: different colonies from soft agar plate(exposed). (a,b) ORF-2; (c,d) b-actin.
37
Ultraviolet-induced transformation of keratinocytes
to enhance LINE-1 activity in embryonal carcinoma
cells (27). We have shown that, of the multiple
exposed populations, only the malignant population,
which formed colonies on soft agar, showed increase
in LINE-1 gene expression as compared with the non-
malignant population. Thus UV enhances LINE-1
activity, which in turn may cause alterations and
modifications in the genome eventually leading to
malignancy. Expression of LINE-1 and ORF-2 thus
can be used as a potential marker to probe UV-
induced malignancy. Its implication in chemically
induced malignancy is under investigation.
We have recently cloned one gene from the
magnetically purified non-apoptotic population (UV
was used to induce apoptosis) from the UV-exposed
apoptotic cells (28). This population being non-
apoptotic, it has a tendency of transformation and
showed upregulation of LINE expression (data not
shown). It is possible that this population (UV
exposed but resistant to apoptosis) may be trans-
formed toward malignancy once exposed to multiple
UV doses. Recently, It has been shown that ZnCl2 can
induce syrian hamster embryo cell transformation
(29). Although ZnCl2 is known for its anti-apoptotic
effect, the cell that survived has a high possibility of
transformation toward malignant phenotype. This
data is consistent with our findings.
It can be summarized that UVB can induce
transformation of human keratinocytes involving
LINE-1. Those cells, which can escape apoptotic
death by UV, are more resistant to UV-induced death
and possibly are more prone to malignant transfor-
mation using LINE-1 RT.
The presence/involvement of other genes in the
elicitation of malignancy as well as possibility of using
them as a predictive tool for carcinogenesis is under
investigation.
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Accepted for publication 15 September 2004
Corresponding author:
Gautam Banerjee
Cell and Molecular Biology
Toxicology Section
Environmental Safety Laboratory
Hindustan Lever Research Centre
B.D. Sawant Marg, Andheri
Mumbai
India
Tel: 191 22 2827 6370
Fax: 191 22 2836 3680
e-mail: [email protected]
39
Ultraviolet-induced transformation of keratinocytes