the molecular basis for cell cycle delays following ... advance/journals/radion/ecco37.pdfthe...

ELSEVIER SCIENCE IRELAND Radiotherapy and Oncology 31 (1994) I - 13

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The molecular basis for cell cycle delays following ionizing radiation: a review

Amit Maity”, W. Gillies McKennaa, Ruth J. Muschel*b ‘Department of Radiation Oncology. hDeparrment of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine,

Philadelphia, PA 19104. USA

(Received 15 September 1993: revision received 8 December 1993: accepted 20 December 1993)

Abstract

Exposure of a wide variety of cells to ionizing (X- or y-) irradiation results in a division delay which may have several components including a G, block, a G2 arrest or an S phase delay. The G, arrest is absent in many cell lines, and the S phase delay is typically seen following relatively high doses (> 5 Gy). In contrast, the G2 arrest is seen in virtually all eukaryotic cells and occurs following high and low doses, even under 1 Gy. The mechanism underlying the G2 arrest may involve suppression of cyclin Bl mRNA and/or protein in some cell lines and tyrosine phosphorylation of ~34”~“’ in others. Similar mechanisms are likely to be operative in the G2 arrest induced by various chemotherapeutic agents including nitrogen mustard and etoposide. The upstream signal transduction pathways involved in the G2 arrest following ionizing radiation remain obscure in mammalian cells; however, in the budding yeast the rud9 gene and in the fission yeast the chkl/rad27 gene are involved. There is evidence indicating that shortening of the G2 arrest results in decreased survival which has led to the hypothesis that during this block, cells repair damaged DNA following exposure to genotoxic agents. In cell lines examined to date, wildtype ~53 is required for the G, arrest following ionizing radiation. The gudd45 gene may also have a role in this arrest. Elimination of the G, arrest leads to no change in survival following radiation in some cell lines and increased radioresistance in others. It has been suggested that this induction of radioresistance in certain cell lines is due to loss of the ability to undergo apoptosis. Relatively little is known about the mechanism underlying the S phase delay. This delay is due to a depression in the rate of DNA synthesis and has both a slow and a fast component. In some cells the S phase delay can be abolished by staurosporine, suggesting involvement of a protein kinase. Understanding the molecular mechanisms behind these delays may lead to improvement in the efficacy of radiotherapy and/or chemotherapy if they can be exploited to decrease repair or increase apoptosis following exposure to these agents.

Key words: Radiation; Cell cycle delay; Cyclin ~53; cdc2

1. Background

Irradiation of a wide variety of cells, including amoeba [l], sea urchin eggs [2], yeast [3], and mammalian cells, causes a division delay. In mammalian cells, this delay may have several components including a G1 arrest, an S phase delay and a G2 arrest. Although this

* Corresponding author, Room 520, Clinical Research Building, University of Pennsylvania, 422 Curie Blvd., Philadelphia, PA 19104, USA.

has heen known for many years, it is only recently that progress in our understanding of cell cycle regulation through checkpoints [4] and feedback controls [5,6], has offered new insight into the underlying biochemical mechanisms. This review will summarize our current knowledge regarding the S phase delay following radiation, the role of ~53 in the G1 arrest and the role of cyclins and ~34’~‘~ in the G2 arrest.

Much of the earlier work on radiation effects on mammalian cells was done in HeLa cells. By calculating the percentage of mitotic figures (mitotic index) at

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A. Maity et al. / Radiother. Oncol. 31 (1994) 1-13

various times, Painter and Robertson [7] showed that in HeLa S3 cells there was a division delay within 2 h following exposure to 500 R which lasted for at least 8 h and disappeared by 14.5 h. By pulse labelling with [3H]thymidine and determining the percentage of labelled cells, they demonstrated an accumulation of cells in S phase between 4 and 8 h following this dose. Painter [8] subsequently showed that this accumulation resulted from a direct depression in the rate of DNA synthesis while the rate of entry from Gt to S remained un- changed.

Yamada and Puck [9] irradiated HeLa S3 cells with lower doses of radiation (0.34-l .35 Gy). They continuously labelled cells with [3H]thymidine for various lengths of time immediately following irradiation. By plotting the percentage of labelled nuclei versus time, they showed that the duration of S phase was not significantly affected by these doses of radiation, nor was there a block from G, to S phase. In contrast, they saw a drop in the frequency of metaphase within 1 h of irradiation followed by a rise, an overshoot and then return to baseline. The amount of time for the relative metaphase frequency to return to baseline ranged from 2 h for 0.34 Gy to 11 h for 1.35 Gy. Since the frequency started to drop within 1 h after irradiation, they inferred that a block must be occurring within 1 h prior to metaphase, most likely late in G2. They directly demonstrated a G2 block by continuously labelling cells with [3H]thymidine and plotting the percentage of labelled metaphases as a function of time. Using this approach, they found that the duration of G2 delay increased proportionately with dose.

After irradiating HeLa S3 cells synchronized by mitotic shakeoff with 3 Gy during different phases of the cell cycle, Terasima and Tolmach [lo] found that the total division delay was minimal after irradiation in G,, maximal for cells in G2 and intermediate for cells irradiated in S. Cells irradiated in G, experienced a slightly prolonged S phase and a minimally prolonged G2 phase. Irradiation in S or G2 resulted in a significant Gz delay. Cells irradiated in S phase also had a prolonged S phase and a decreased rate of DNA synthesis. No significant G, delay was seen for HeLa cells irradiated in any phase.

Yu and Sinclair [ 1 l] found that synchronized Chinese hamster V79 cells showed a dose dependent mitotic delay following 2.35-7.1 Gy which was greatest for cells irradiated in late S phase, least for cells in Gi and intermediate for cells in G2. Likewise, Leeper et al. [12] showed that CHO (Chinese hamster ovary) cells synchronized by mitotic shakeoff also exhibited a significant division delay following 1.5-6 Gy which was dose- and cell-cycle-dependent. The later in the cell cycle irradiation occurred, the greater was the delay (minimal for G, cells; maximal for late S/G2 cells). As with HeLa cells, most of the delay was due to a block in Gz, but

less pronounced delays in progression through Gi or S were detected [ 131.

Similar effects have been observed in mouse L-cells. Whitmore et al. [14] showed that there was a significant division delay after 20 Gy (19 h) or 50 Gy (25 h) which was primarily due to a reversible block in GZ. By using various radiolabelling techniques, Mak and Till [ 151 were able to detect a decrease in the progression of cells from G, to S and a decrease in the rate of DNA synthesis with doses as low as 2.2 Gy and 5.8 Gy, respec- tively.

Work has also been done on cells taken directly from human tissue. Lajtha and coworkers [ 16,171 showed that in human bone marrow cells, doses between 5 and 50 Gy resulted in a decrease in the rate of DNA synthesis as well as a block in movement of cells from G, to S whereas doses of 2 and 3 Gy caused only the latter. Little [18] performed experiments on diploid human amnion cells taken from placenta which had an extremely slow turnover. By continuously labelling with [ 14C]thymidine for several days and determining the labelling index as a function of time, he showed that even doses as low as 0.1 Gy decreased the flow of cells from Gi to S. This decrease was dose-dependent from 0.1 to 3 Gy with an almost complete Gi arrest at 3 and 10 Gy.

It has been known for many years that caffeine can partially abolish the division delay induced by radiation. Treatment with l-2 mM caffeine prior to irradiation reduces both the inhibition of DNA synthesis (S phase delay) and the G2 delay in both CHO [19] and HeLa cells [20]. Boynton et al. [21] showed that S-180 ascites tumor cells grown intraperitoneally in mice underwent a G2 block following whole body irradiation which could be reduced by pretreating the mice with caffeine.

2. G2 arrest in mammalian cells

Many years ago our laboratory became interested in the molecular basis for radiosensitivity and decided to use as a model system rat embryo libroblasts transfected with various oncogenes. Several lines were created [22] by transfecting with H-rus and myc (2.8, 2.10 and 3.7) or with myc alone (Myc-Ret and MR4). All three lines transfected with H-rus and myc were significantly more radioresistant (Do, 1.68-2.17) than the two transfected with myc alone (De, 1.06- 1.08) which had a sensitivity comparable to the parental cells. The difference in radiosensitivity between 3.7 and MR4 was not due to differences in the induction of double strand breaks or their rejoining [23]. However, the radioresistant lines displayed a much greater Gz delay following irradiation than the sensitive lines.

Su and Little [24] have shown a similar correlation in human diploid cells transfected with SV40 large T antigen. The SV40 immortalized lines were more radioresistant than the parental lines (Do, 1.65-2.33 vs.

A. Maity et al. /Radiother. Oncol. 31 (1994) 1-13 3

1.20-1.35) and showed a greater G2 delay following doses ranging from 2 to 8 Gy.

Since making the correlation between radiosensitivity and G2 delay in this rat embryo model system, our laboratory has been trying to determine the mechanisms underlying the radiation-induced G2 arrest. In order to describe these studies, it is necessary to first summarize our current knowledge regarding the biochemistry of the G,/M transition (for review see Ref. 25).

2.1. Cyclins, cdc2 kinase and the G/M transition

Entry of cells into mitosis is regulated by mitosis pro- motion factor (MPF), a complex of two proteins, p34’d’2 and cyclin B. In its activated form, MPF phosphorylates key proteins such as histones and nuclear lamins, which is thought to lead to the events characterizing mitosis such as chromosome condensa- tion and dissolution of the nuclear envelope. In vitro, MPF activity can be assayed by measuring phosphorylation of histone Hl . In order for cells to complete mitosis and enter Gi, cyclin B must be degraded [26]. Beach et al. [27] originally isolated ~34’~~’ from a temperature sensitive mutant of the fission yeast Schizosaccharo- myces pombe which at the restrictive temperature arrested in either G, or late G2. They also isolated the homologous gene in the budding yeast Saccharomyces cerevisiae, cdc28, previously described by Hartwell et al. [28], as well as the human homolog [29]. The cdc2 gene was found to encode a 34-kDa phosphoprotein with kinase activity, often referred to as ~34’~” or simply p34. p34cdc2 has subsequently been shown to be essential for the G2/M transition in mammalian cells, but its role in Gi is less defined. Riabowol et al. [30] showed that inhibition of ~34’~‘~ in rat fibroblasts by microinjection of antibodies directed against it prevented entry into mitosis.

The cyclins were discovered by Hunt et al. in marine invertebrates as proteins whose levels oscillated with the cell cycle [31]. Two proteins were identified with slightly different molecular weights and temporal patterns of expression; the one whose level rose earlier and fell earlier was called cyclin A and the other cyclin B. These origi- nal cyclins were mitotic cyclins involved in the Gz/M transition; however, subsequently, Gi cyclins have been discovered (for review see Ref. 32).

Cyclins have been cloned from diverse species including Schiz. pombe [33], humans [34], Drosophila [35], alfalfa [36] and rodents [37]. Sequence comparison shows that there is a relatively conserved region called the ‘cyclin box’ located in the middle third of the protein. Motifs within this can be used to separate the A- from B-type cyclins. All eukaryotes examined to date have been found to contain B-type cyclins; however, A- type cyclins have not been found in yeast. There are at least two types of B cyclins, Bl and B2, in Xenopus [38]

and four types in Sac. cerevisiae [39], but the distinction between these is unknown.

The function of cyclin A is less well defined than that of cyclin Bl. Cyclin A appears to have an important role in DNA synthesis as well as in the G2/M transition. In rat embryo Iibroblasts [40] and HeLa cells, microinjection of anti-cyclin A antibody in G, inhibited DNA synthesis. Microinjection of this antibody into HeLa cells in G2 halted progression into mitosis [41]. Consis- tent with these findings, immunofluorescence in primary human fibroblasts shows that cyclin A has a predominantly nuclear localization from S phase on- ward whereas cyclin Bl accumulates in the cytoplasm of interphase cells and only enters the nucleus at the begin- ning of mitosis [42]. Also consistent with its involvement in DNA synthesis, in synchronized HeLa cells, transcription of the cyclin A gene starts about 2 h before cyclin Bl [43]. Whereas in HeLa cells, the level of cyclin Bl protein rises in S phase, peaks in early M phase and falls abruptly at the metaphase/anaphase transition [34], the level of cyclin A starts to rise earlier, peaks earlier and falls abruptly in metaphase [43]. Likewise, the histone Hl kinase activity of cyclin A occurs earlier than that of cyclin B 1. The exact function of cyclin A in DNA synthesis is unknown; however, it appears to be carried out in complex with ~33’~~, a protein related to ~34’~” which can associate with cyclin A but not cyclin B 1 [44]. There is evidence that the cyclin Alp33caJ complex binds to and phosphorylates ~107, an Rb-like protein [45]. These three proteins have been found in a quater- nary complex with the transcription factor E2F in S phase [46]. Since E2F binding sites are found within promoters of many genes involved in DNA synthesis, such as thymidine kinase and dihydrofolate reductase, this suggests how cyclin A may be involved in DNA synthesis.

2.2. Regulation of cdc2 kinase by phosphorylation

The association of ~34’~” with cyclin B is necessary but not sufficient for generation of kinase activity. There are several residues within ~34’~” whose phosphorylation modifies its activity. In Schiz. pombe, it has been shown that Tyrl5 of ~34’~~~ is phosphorylated in late Gz but must be dephosphorylated in order for cells to proceed into mitosis [47]. Thr167 is also phosphorylated, but this activates MPF [48].

In mammalian cells, the phosphorylation of ~34’~‘~ is also important in regulating its activity. Norbury et al. [49] and Krek and Nigg [50] showed that for both mouse and chicken ~34 cdc2 there exists a Thr14 as well as a Tyrl5 whose phosphorylation negatively regulates kinase activity. Both residues become dephosphorylated as cells enter mitosis. In mammalian ~34’~‘~, Thr167 is

4 A. Maity et al. / Radiother. Oncol. 31 (1994) 1-13

the homolog of the Schiz. pombe Thr161 and regulates kinase activity via phosphorylation [51-531.

The exact timing of the phosphorylation of these residues on ~34’~‘~ relative to the binding of cyclin Bl to p34cdc2 is at present uncertain. It appears that phosphorylation of Tyr15 and Thr14 requires cyclin binding [54]. Results in Schiz. pombe [48] and human ~34’~‘~ [51] suggest that cyclin binding occurs after Thr161/167 is phosphorylated; however, other data suggest that phosphorylation occurs after cyclin binding [53,55]. One model [55] for mammalian cells is that cyclin B binds to unphosphorylated ~34’~” in S phase and induces the phosphorylation of all three residues. The p34cdc2/cyclin Bl complex is kept inactive until Thrl5 and Thr14 are dephosphorylated at the G2/M transition at which time the complex is activated. At the metaphase/anaphase transition, cyclin Bl is degraded and Thrl61 on ~34’~” is dephosphorylated.

A number of regulators of ~34’~‘~ phosphorylation have been identified including the phosphatase cdc25 and the kinases wee1 and mikl. cdc25 was originally cloned by Russell and Nurse [56] from Schiz. pombe, and several years later the human homolog was cloned [57]. cdc25 encodes a phosphatase which specifically dephosphorylates Tyrl5 in lower eukaryotes and both Tyrl5 and Thr14 in higher eukaryotes [58,59]. The phosphatase activity of cdc25 is itself regulated by phosphorylation, increasing immediately prior to mitosis [60]. Hoffman et al. [61] showed that in human cells cdc25 is hyperphosphorylated near mitosis at the time of maximal phosphatase activity. They also showed that this form can activate the p34cdc2/cyclin Bl complex. Interestingly, this activated complex can phosphorylate cdc25, raising the possibility of an autocatalytic feedback loop.

Another mitotic regulator, wee1 was cloned by Russell and Nurse [62] by complementation of a yeast mutant that entered mitosis prematurely. This gene encodes a 107-kDa dual specificity protein which can phosphorylate both tyrosine and serine residues [63]. The human homolog of wee1 has been cloned [64] and found to encode a 49-kDa protein. Parker and Piwnica- Worms [65] showed that in vitro, human p49’““’ catalysed the phosphorylation of Tyr 15 but not Thr14 of p34cdc2/cyclin Bl. This resulted in an inhibition of histone Hl kinase activity which was partially prevented by the addition of cdc25. McGowan and Russell [66] also have shown that wee1 can phosphorylate Tyrl5 but not Thr14 of ~34’~” in the MPF complex. Furthermore, overexpression of human wee1 in HeLa cells by transient transfection led to an arrest in G2 without affecting the increase in cell volume. In Sac. cerevisiae, wee1 activity is negatively regulated by phosphorylation at its C-terminus end by another kinase, niml (new inducer of mitosis)/cdrl (changed division response) [67].

A second gene, mikl (mitosis inhibitory kinase) was cloned by Lundgren et al. [68] by complementation of another Schiz. pombe mutant which also entered mitosis prematurely. These investigators showed that wee1 and mikl have redundant functions. Deletion of both resulted in tyrosine dephosphorylation of ~34’~‘~ and premature lethal entry into mitosis.

Both cdc25 and wee1 are thought to be important in coupling mitosis and cell division to earlier events in the cell cycle. Schiz. pombe mutants which overexpress cdc25 undergo premature cell division, resulting in cells smaller than wildtype. Hydroxyurea, which inhibits DNA synthesis, halts cell cycle progression in wildtype cells; however, in these cdc25 overexpressors, cell division continues, resulting in anucleate cells or cells containing fragments of chromosomal material [69]. In contrast, weel-50 mutants which lack functional wee1 and also divide prematurely are arrested following exposure to hydroxyurea. Based on these results, it has been suggested that cdc25 but not wee 1 is important in coupling cell division to the completion of DNA replication. Because overexpression of wee1 results in a dose- dependent delay in mitosis until cells have grown to a larger size than normal, it was thought that perhaps its function might be to coordinate cell division to cell size. More recent work [70] suggests that this model may be too simplistic as there are situations in which the DNA replication checkpoint is still intact in spite of the absence of cdc25 and other instances in which overexpression of wee1 can restore it.

2.3. Role of cyclins and cdc2 kinase in G2 arrest

Because cyclin Bl is required for the G2/M transition, our laboratory decided to examine the effects of irradiation on its mRNA and protein [71]. HeLa cells were originally chosen for the study because at the time these investigations were initiated, the human cyclin Bl cDNA had been cloned [34]. HeLa cells were synchronized at the G&S boundary using a sequential thymidine/aphidocolin block. Shortly after release from the block, the cells were irradiated with 10 Gy, and samples were taken at serial timepoints from replicate plates for flow cytometry and RNA analysis. The irradiated cells showed a delayed entry into Gz compared with control unirradiated cells, consistent with the previously described S phase delay 171. However, once in Gz, the irradiated cells stayed for at least 9 h longer than the controls. The interesting observation is that cyclin Bl mRNA levels remained low during this accumulation in GZ. In contrast, in the control cells, cyclin Bl mRNA rose ten-fold in G2 and then fell as cells progressed into Gt. This led to the hypothesis that suppression of cyclin Bl mRNA was partly responsible for the Gz arrest after irradiation in S phase. Another

A. Muiry el al. / Rudiotlwr. Oncol. 31 (1994) 1-13

experiment was performed in which HeLa cells were synchronized in G,/S, released and then irradiated with 6 Gy in late S/early G,. Again, the irradiated cells showed a G2 delay, taking 7.5 h to enter Gi compared with 2.5 h for the control cells. Northern blotting showed that the cyclin Bl mRNA did rise and peak in a pat- tern similar to that seen in control cells. However, Western blotting showed that the level of cyclin Bl protein was suppressed for 4-5 h following radiation. In contrast, in the control cells, cyclin Bl protein peaked within 1 h of time zero and then fell within 3 h, corresponding to an exit of cells into Gi. Therefore, radiation appears to be able to downregulate cyclin Bl expression in HeLa cells at the mRNA or protein level depending on the phase of the cell cycle in which they are irradiated.

In more recent experiments, Muschel et al. [72] have shown that the suppression of cyclin Bl mRNA with irradiation of HeLa cells in S phase is dose dependent and can be detected after as little as 2 Gy. Using similar syn- chrony experiments as described above, the effects of radiation on cyclin A mRNA and protein were also analysed. In contrast to cyclin Bl, cyclin A mRNA started to increase in irradiated cells at the same time as in control cells. As cells accumulated in G2/M in the irradiated cells, the cyclin A mRNA level continued to rise and peaked at a level higher than that seen in control cells. Likewise, the cyclin A protein levels also over- shot the levels seen in control cells. It is unknown whether this overshoot is related to the supression of cyclin B or whether it is part of the mechanism of the Gz delay.

Datta et al. [73] examined the effects of radiation on cyclin Bl expression using a human myeloid leukemia cell line, U-937. They found that a population of cells enriched in G, by elutriation when irradiated with IO Gy showed a decrease in cyclin B mRNA expression with time but a progressive increase in cdc2, cyclin A and cdc25 mRNA expression. A similar decrease in cyclin Bl mRNA expression was seen following irradiation of S phase enriched cells with 10 Gy.

Lock and Ross [74] investigated the effects of radiation on CHO cells and found that exposure of asyn- chronously dividing cells to 8 Gy resulted in an accumulation of cells in Gz and a rapid inhibition of P34’Jc2 histone Hl kinase activity within I h. Uckun [75] found that irradiation of Ramos Burkitt’s lymphoma and K562 erythroleukemia cells with l-8 Gy resulted in enhanced tyrosine phosphorylation of P34 cJc2 and an inhibition of its histone Hl kinase activity which correlated temporarity with the Gz delay. Furthermore, pretreatment with herbimycin. a known inhibitor of protein tyrosine kinases, prevented this inhibition of kinase activity as well as the G, delay.

As mentioned previously, caffeine has long been

s

known to abolish both the S phase delay and the G? arrest induced by radiation. Hain et al. [76] showed that in V79 hamster cells, incubation in 4 mM caffeine 4 h after exposure to 7 Gy reversed the accumulation of cells both in S and Gz phase. An explanation of this was suggested by the fact that ~34”~“’ kinase activity increased by a factor of four within 30 min of caffeine treatment.

A number of chemotherapeutic agents can also induce a Gz delay. Lock and Ross [74] found that CHO cells exposed to the epipodophyllotoxin etoposide underwent a Gz delay during which ~34”“~ histone Hl kinase activity was inhibited. More recently, Lock [77] has shown that the cells arrested in Gz following exposure to etoposide had a persistent tyrosine phosphorylation of ~34’~” which could explain their loss of histone Hl kinase activity. A similar correlation between G2 delay and persistent tyrosine phosphorylation of ~34”‘“” has been observed in HeLa cells following exposure to the drug camptothecin, a DNA topoisomerase I inhibitor [78], and in CA46, a human lymphoma line, following exposure to nitrogen mustard [79]. The persistent hyper- phosphorylation of ~34”“” in human lymphoma cells after exposure to nitrogen mustard is accompanied by a failure of cdc25 to be converted to its active hyperphosphorylated form [80]. O’Connor et al. [81] recently showed that the nitrogen mustard-induced G2 delay in CA46 cells correlates with a suppression of cyclin Bllp34”““’ and cyclin Alp34”““’ kinase activity but not cyclin A/p33cdk2 kinase activity. Furthermore, pentoxifylline, a methylxanthine related to caffeine which has been shown to enhance cytotoxicity of alkylating agents [82], abolished the delay and reverted the cyclin A and Bl kinase activity to that seen in controls.

It has been hypothesized that the Gz arrest following DNA damage may also serve the function of allowing cells to repair damage. Tobe.y [83] speculated that ‘a surveillance mechanism operates throughout G, to eliminate from the proliferative mode cells with altered DNA. Such a surveillance mechanism would examine cells at multiple stages within G, to ensure that either the damaged DNA was repaired normally, in which case a cell would be returned to cycle, or a unrepaired cell would be converted to a non-viable state’. There is no direct evidence for this hypothesis; however, there is some circumstantial evidence. As discussed above, investigators have made correlations between the length of G2 and radiosensitivity [22,24]. Caffeine, which abrogates the G2 delay following radiation, decreases cell survival (841 although this is difficult to interpret since the drug has many effects. Likewise, pentoxifylline has also been shown to sensitize several cell lines in culture to radiation [85].

Arrest in G2 following exposure to radiation and various other DNA damaging agents is seen in diverse

6 A. .&laity et al. / Radiother. Oncol. 31 (1994) I-13

cell types; however, the actual mechanism may vary. In irradiated HeLa cells, the Gz arrest has been associated with downregulation of cyclin B at either the RNA or protein level. However, in other systems using different cells and/or agents, alterations in ~34’~‘~ phosphorylation have been described. All of these changes have a common endpoint: the inactivation of MPF, which prevents entry into mitosis. The basis for these variations remains unclear. It is possible that the same cell type may use different mechanisms depending on the agent to which it is exposed. For example, in HeLa cells, changes are seen in cyclin B expression following radiation but in ~34’~‘~ phosphorylation following exposure to camptothecin. It is noteworthy that some of the cell types on which these studies have been performed are derived from lymphocytes, which, unlike most cells, undergo a mitotic death mediated by apoptosis rather than a postmitotic interphase death. It is not known whether this has any bearing on the mechanism of G2 delay.

It is clear we have just started to outline the mechanisms responsible for the Gz arrest following DNA damage. It needs to be established whether DNA damage is actually repaired during the Gz arrest and whether modulation of the duration of this arrest can alter cell viability. The details regarding detection of this damage and transduction of this signal to ultimately inactivate MPF are also obscure. Since it appears that different pathways can be used to achieve the same end, it remains to be determined how cells choose a certain pathway over another.

3. G2 arrest in yeast

Yeasts, which have proven so useful in understanding the genetics of cell cycle regulation, have also been helpful in gaining insight into the genetic basis for the G, delay following radiation. Much of the pioneering work in this area was done by Weinert and Hartwell [86] who focused their attention on the budding yeast Sac. cerevisiae in which individual cells can be followed easily through the cell cycle by changes in nuclear morphology. They isolated a particular mutant, rad9, which lacked the ability to arrest in G2 following X- irradiation. A dose of 20 Gy, which killed 68% of raa’!9 mutants, caused G2 arrest in only 18% of the cells. The same dose killed 52% of RAD+ (wildtype) cells but arrested 49% of the cells. They then performed experiments with yeast cells synchronized in G2 by using the microtubule blocking agent methylbenzamide-2-y-l- carbamate (MBC). They irradiated the cells with 0, 20 or 80 Gy, removed the MBC and replated the yeast on fresh plates. RAD+ cells showed a dose-dependent division delay which was much greater than that seen in rad9 mutants. They then irradiated cells synchronized in G, with MBC and left them in the drug in order to

impose a drug-induced delay. rad9 mutants held in MBC for 4 h after irradiation showed dramatic improvement in cell viability, not seen in RAD+ cells.

Weinert and Hartwell [87] subsequently cloned the ra& gene by complementation and found it to encode a protein with no similarity to any known proteins. rad9 mRNA is constitutively expressed by cells and not induced by irradiation. A ra&null mutant which com- pletely lacked the gene did not display a Gz arrest following irradiation. However, in the absence of irradiation, this mutant exhibited normal growth and cell cycling although it had a spontaneous rate of chromosomal loss and recombination 7-21-fold greater than the wildtype. Based on these results, Weinert and Hart- well have proposed that rad9 performs a surveillance function along the lines of Tobey’s hypothesis [83] checking on DNA integrity and signalling the cell to arrest in G2 in the event that damage is detected.

Weinert [88] has isolated five other genes, radl7, rad24, and mecl-mec3 (mitosis entry checkpoint) which are required for G2 arrest following radiation. mecl and mec2 also appear to be important in sensing unreplicated DNA since in their absence, cells fail to arrest following exposure to hydroxyurea.

Much work has also been done on the fission yeast Schiz. pombe in which the septation index (% of cells with forming septa) can be used as a measure of division delay. Rowley et al. (891 used the weel-50 mutant which at the restrictive temperature lost wee1 function. At the permissive temperature, these mutants behaved like the wildtype following irradiation with a transient decrease in the septation index; however, at the restrictive temperature, there was no division delay. Furthermore, this loss of Gz delay was accompanied by poorer survival, supporting the notion that the G2 delay is important for DNA damage repair and viability. In contrast, a mutant which lacked functional mikl, which, as discussed previously, has overlapping function with wee1 [68], did not lose the radiation-induced G2 delay. The investigators also irradiated mutants which overexpressed cdc25. As expected, these cells also displayed a reduced G2 delay compared with the wildtype. Further differences between these mutants were highlighted by exposure to hydroxyurea. As shown previously by Enoch and Nurse [69], weel-50 mutants underwent cell cycle arrest following exposure to hydroxyurea; however, the mutants overexpressing cdc25 showed no delay in cell cycle progression, and mikl-null mutants showed a reduced delay. This suggested that wee1 and mikl operate in two different pathways, the former monitoring radiation-induced DNA damage and the latter monitoring DNA replication. However, wee1 may not be essential for the radiation-induced G2 arrest in all Schiz. pombe since weel-null mutants have been recently described which can still arrest following DNA damage El.

A. Maity et al. / Radiother. Oncol. 31 (1994) l-13

Other genes have been identified in Schiz. pombe that appear to function as checkpoints following UV irradiation, specifically radl, 3,9 and 17 [90]. Yeast containing mutations of any of these four genes fail to arrest after exposure to either UV irradiation or hydroxyurea and display an extreme sensitivity to both. radl and rad3 have also been shown to be necessary for the G2 arrest following y-irradiation [9 1,921. By screening Schiz. pombe for hydroxyurea sensitivity, Enoch et al. [70] identified five other genes, husl-hus5 (hydroxyurea sensitive) which also appear to function as checkpoints. Mutants of all five of these also showed extreme sensitivity to W irradiation. The relationship between these nine genes is unknown; however, it is hypothesized that they act in a pathway upstream of weel, mikl and cdc25, monitoring both DNA replication and damage.

Recently Walworth et al. (931 isolated a gene, chkl (checkpoint kinase), from Schiz. pombe which encodes a putative serine/threonine kinase and whose overexpression in wildtype yeast caused a mitotic delay. Disruption of this gene causes failure to arrest in G2 following UV irradiation, rendering cells extremely radiosensitive. These chkl-disrupted mutants do arrest after exposure to hydroxyurea, indicating that this gene is not involved in the pathway sensing DNA replication. Carr et al. (un- published data) independently isolated a gene from Schiz. pombe whose deletion results in loss of the G2 checkpoint following ionizing radiation. This gene was named rad27; however, it has since been shown to be identical to chkl. Based on these observations, it has been suggested that in Schiz. pombe, chkllrad27 couples the DNA damage sensing pathway to the cdc2- dependent cell cycle pathway.

The work done in yeast supports the idea that the G2 delay following radiation is an important determinant in cell survival. In both the fission and budding yeast, mutants which are unable to arrest in Gz consistently show a poorer survival following radiation than the wildtype. In some respects, more progress has been made in defining the genetics of the G2 delay in yeast than in mammalian cells. This is to be expected given the relative ease of genetic manipulation in the former compared with the latter. It is hoped that many of the yeast genes will turn out to have structurally or functionally homologous counterparts in mammals so that complementation or hybridization techniques can be used to isolate the corresponding mammalian genes.

4. Gl arrest

As discussed in section 1 above, a G1 arrest is not seen in all cell lines following radiation. Recent work suggests that its presence depends on the ~53 status of the cell line. Kastan et al. [94] showed that irradiation of ML-l myeloblastic leukemia cells with 0.5-4 Gy caused an accumulation of cells in both Gs,t and G2/M,

1

consistent with two different blocks. The G, arrest predominated at lower doses and the G2 arrest at higher doses. Cells irradiated in S phase were able to progress into Gz/M. Because of the known anti- proliferative effects of ~53 which will be further described below, these investigators sought to determine whether there was any correlation with changes in its expression. Indeed, the ~53 protein level rose l-2 h after irradiation and required 48-72 h to return to normal. Northern blots showed no change in ~53 mRNA, suggesting that the elevation in protein levels was due to a posttranslational increase in stability.

The responses of a number of other hematopoietic 1941 and non-hematopoietic [95] cell lines (human libroblasts, colorectal carcinoma cells, osteosarcoma cells) were noteworthy. In general, cell lines which, like ML- 1, express wildtype ~53 exhibited a radiation-induced G, arrest but not lines expressing mutant ~53 or lacking ~53 altogether. In contrast, the G2 arrest following irradiation remained intact regardless of ~53 status. Further- more, transfection of mutant ~53 into RKO cells which express wildtype ~53 attenuated the Gi arrest. Con- versely, transfection of wildtype ~53 into HL60, a cell line lacking ~53 and not showing radiation-induced G, arrest, resulted in a partial restoration of the arrest.

More recently, Kastan et al. (961 have investigated the relationship between ~53 and radiation-induced arrest using fibroblasts from ~53 ‘knockout’ transgenic mice in which both wildtype pS3 genes have been deleted using the technique of homologous recombination. These mice develop normally except for an increased frequency of certain tumors, specifically lymphomas and sarcomas [97]. Fibroblasts cultured from these mice were used in experiments in early passage while they still had a normal karyotype and morphology. Fibroblasts from normal mice with both intact ~53 genes showed a marked G, arrest after 2 or 4 Gy. In contrast, libroblasts from mice lacking both ~53 genes showed no arrest, and libroblasts from mice with only one intact ~53 gene showed an intermediate response.

The authors then investigated the possible role of gadd45 in the G, arrest. The gadd (growth arrest and DNA damage-inducible) genes are a set of five genes [98] whose transcription in CHO cells is markedly increased by exposure to DNA damaging agents such as the alkylating agent methylmethanesulfonate (MMS) or by growth cessation signals such as serum starvation. One of these genes, gadd45, has been found in human cells and is distinct from the other gads genes in that it can also be strongly induced by X-irradiation [99]. Doses as low as 2 Gy cause a 3-4-fold increase in gadd45 mRNA several hours following irradiation. Kastan et al. [96] found an excellent correlation between gaaV45 mRNA induction and ~53 status. Cell lines containing both intact ~53 genes showed a 2-lo-fold increase in gadd45 mRNA 4 h after 20 Gy whereas lines

8 A. Moiry ct ul. / Rudiothur. Onwl. 31 (1994) I-13

with abnormal ~53 function showed no induction during this time period. Cells from patients with ataxia- telangiectasia which lacked the ~53 response to radiation also showed defective induction of g&d45 mRNA. The authors went on to analyse the DNA sequence of gadd45 and found that the third intron of the gadd45 contains a sequence with a high degree of homology to a previously described ~53 binding sequence [ 1001. They showed that an oligomer containing this sequence could bind wildtype but not mutant ~53 and that when this oligomer was placed upstream of a CAT (chloramphen- icol acetyltransferase) reporter gene, transcription could be activated by wildtype ~53. Based on these results, the authors speculated that gadd45 may act downstream of ~5.3 in a signal transduction pathway necessary for the G, arrest, but this remains to be proven.

The relationship between ~53 and the G, arrest has been confirmed by O’Connor et al. [IO11 who found that they could correctly predict ~53 status (wildtype or mutant) in 15 of 17 Burkitt’s lymphoma and lym- phoblastoid lines by determining whether they exhibited an intact G, arrest following 6.3 Gy. The two cell lines which had a reduced ability to arrest in Gi despite both p53 genes being intact turned out by Western blotting to have a blunted induction of ~53 protein following radiation.

These results suggest that many established cell lines fail to show a Gi arrest following radiation because they lack wildtype ~53. For example, HeLa cells, which were originally derived from a human cervical carcinoma, have low levels of ~53 as a result of infection with human papillomavirus which expresses the E6 protein which targets ~53 for destruction [ 1021.

Although the suggestion was made that the G, arrest might be important for enhanced DNA repair, Kastan’s group has been unable to demonstrate that the loss of this arrest increases radiosensitivity. Fibroblasts from ‘~53 knockout’ mice with either one or two copies of the gene deleted showed no difference in radiosensitivity compared with those taken from control littermates, and transfection of mutant ~53 into RKO cells did not alter radiosensitivity [ 1031.

In fact, in some settings, mutant ~53 and loss of G, arrest is associated with increased radioresistance. Lee and Bernstein [104] found that bone marrow cells taken from transgenic mice which contained mutant ~53 displayed &gnificant resistance following -r-irradiation compared with cells from wildtype httermates (D,, 2.48-2.80 vs. 1.75 Gy). O’Connor et al. [ 1011 found that in their lymphoma cell lines, loss of wildtype ~53 and Gi arrest was associated with radioresistance although this was not an absolute correlation.

It is difficult to reconcile these seemingly contradic- tory results; however, an observation made in thymocytes may help to clarify this dilemma. Wildtype thymocytes are exquisitely sensitive to doses as low as I

Gy, showing a dramatic increase in ~53 level within 1 h of radiation and undergoing an apoptotic cell death [105]. In contrast, thymocytes from homozygous null ~53 mice with both copies of the gene deleted are extremely resistant to radiation-induced apoptosis [105,106] with doses as high as 20 Gy in spite of a normal apoptotic response when exposed to glucocortic- oids. Thymocytes from heterozygous mice with one intact and one deleted copy of thep53 gene display intermediate sensitivity.

It has been suggested [103] that in some cell types such as lymphocytes and bone marrow cells, ~53 is important for triggering both apoptosis and a Gi arrest following radiation but that in others such as fibroblasts it is important only in initiating a Gi arrest. In the former case, loss of wildtype ~53 may lead to decreased apoptosis which is manifested as increased radioresistance. However, in the latter case in which the ~53 signal is not important for apoptosis, loss of wildtype ~53 function may not affect radiosensitivity.

The work cited above has been extremely helpful in elucidating the function of ~53 within the cell. In spite of intense investigation for over a decade, surprisingly little is known in this regard. Originally discovered to complex with simian virus 40 (SV40) large T antigen in cells transformed with this virus [ 107,108], wildtype ~53 has been shown to have a potent anti-proliferative effect on cells using a variety of assays. Introduction of wildtype ~53 into rat embryo fibroblasts [log] and cell lines derived from osteosarcomas [l lo], colorectal carcinomas [l 1 l] and glioblastomas [112] arrests cells in G, and prevents entry into S phase. Its introduction into Saos-2, a human osteosarcoma cell line lacking endogenous ~53, decreases tumorigenicity as assayed by subcutaneous injection into nude mice and colony formation in soft sugar [I 131.

Mutations in ~53 have been found in diverse sporadic (non-familial) tumors [114] and in fact are the most common genetic change found in human neoplasias [115]. The Li-Fraumeni syndrome [116] which results in the autosomal dominant inheritance of certain tumors, most notably sarcomas and breast carcinomas, often involves a germ-line mutation in ~53 [117]. Some of these mutant ~53 proteins can act as cooperating oncogenes, that is when cotransfected into rat embryo tibroblasts with the rus oncogene, they greatly increase the efficiency of transformation.

There is mounting evidence that ~53 can function as a transcriptional factor [118-1201. More recently, investigators have determined that wildtype ~53 can bind to specific DNA sequences in vitro [ 100,121,122]. Templates containing multiple copies of a 33-bp ~53 binding elements upstream of a CAT reporter gene can be transactivated by wildtype ~53 but not mutant ~53, both in vivo [ 1231 and in vitro [ 1241. Furthermore, addition of mutant ~53 can inhibit transactivation by

A. Muity em al. / Radiorhur. Oncol. 31 (1994) 1-13 9

wildtype ~53. In spite of the ability of ~53 to activate transcription of genes in experimental constructs, only a few genes have been found to contain naturally occurring ~53 binding sequences: the muscle creatine kinase gene [125], the gadd45 gene [96] and the mdm2 gene [126]. In fact, ~53 has been shown to repress transcription from many more genes including c-fos, c+n, fl- actin, hsp 70, interleukin 6 and ~53 itself [127,128] through an unknown mechanism.

The ~53 protein does not seem to play a critical role in normal cell growth and differentiation since ‘knockout’ mice in which both wildtype genes have been deleted develop normally [97]. However, these mice do develop certain types of tumors prematurely. This may be related to the observation that in some model systems loss of ~53 can lead to genomic instability [ 129,130]. Normal diploid libroblasts [129] undergo cell cycle arrest when exposed to the drug N-(phosphonacetyl)-L- aspartate (PALA) and display an undetectable level of gene amplification (< 109). In contrast, fibroblasts from ~53 ‘knockout’ mice in which both genes are deleted continue to cycle and display a high level of gene amplification upon exposure to PALA. Taking these findings together with the results relating to radiation, one could speculate that ~53 does not play an important role in the normal day-to-day activity of the cell. How- ever, in the’ presence of damage caused by agents such as radiation, ~53 may be important in either signalling cells to undergo apoptosis or repair the damage in order to prevent genomic instability and subsequent tumori- genesis. Thus, ~53 may serve as a ‘guardian of the genome’ [ 13 I] or ‘molecular policeman’ [ 1321. Although an interesting hypothesis, it remains unproven. Also, it does not explain why in those cell lines in which the ~53 signal initiates a G, arrest but not apoptosis following radiation, loss of wildtype ~53 function does not seem to increase radiosensitivity.

5. S phase delay

Irradiation also causes a slowing of progression through S phase, felt to be due to a depression in the rate of DNA synthesis. This depression of DNA synthesis displays a biphasic dose response with a steep (radiosensitive) component at lower doses and a shallow (radioresistant) component at higher doses. Using autoradiography in the presence of t3H]thymidine, Watanabe [133] was able to measure the length of elongating DNA fibers in mouse leukemia L5178Y cells. He found that the rate of DNA chain growth was reduced linearly when plotted against radiation dose with a Do of 130 Gy. Therefore, he postulated that the shallow (radioresistant) component of the dose response curve was due to inhibition of DNA chain elongation. Subsequently, others have shown that the steep (radiosensitive) component of the dose response curve is

due to inhibition of replicon initiation. Makino and Okada [134] pulse-labelled L5178Y cells with [‘Hlthymidine and then performed alkaline sucrose gradient sedimentation of the DNA. Using this technique, they conclusively showed that 5 Gy caused a decrease in the rate of DNA synthesis. Furthermore, they found a deficiency in smaller sized DNA’s up to 2 h following radiation compared with non-irradiated controls, consistent with an inhibition of replicon initiation rather than chain elongation. The same technique has been used to show that doses from 5 to 8 Gy cause an inhibition of replicon initiation in CHO. HeLa and mouse L cells [135-1371.

Based on estimates of target size, Painter and Young [136] hypothesized that a single radiation hit blocks the initiation of entire clusters of replicons, not just one replicon. Povirk and Painter [ 1381 irradiated HeLa and bovine kidney cells which had been incubated in media containing 5-bromodeoxyuridine to allow for substitu- tion of approximately 20% of the thymine residues with BrdU. They then irradiated the cells with 313 nm light which produces strand breaks with high efficiency in BrdU-substituted DNA but has little effect on un- substituted DNA. Because this led to a suppression of replicon initiation, they inferred that the target responsible was either the DNA itself or something in the imme- diate vicinity.

Wang and Iliakis [ 1391 examined the effect of radiation on DNA synthesis in the oncogene transfected rat embryo fibroblast system described previously. Both the radioresistant 3.7 line (myc and ras transfected) and the radiosensitive MR line (myc transfected) showed the expected biphasic curve when the rate of DNA synthesis was plotted versus dose; however, the inhibition of replicon initiation for the 3.7 cells was much greater than the MR cells. Furthermore, in time course experiments, the inhibition of the rate of DNA synthesis in the 3.7 cells was much more prolonged than that seen in the MR cells. Similar experiments were carried out on lines transfected with other oncogenes and in general, lines transfected with ras and myc or ras and adenovirus Ela showed increased inhibition of DNA synthesis compared with those transfected with myc or Ela alone, leading to the speculation that t-as is involved in the signal transduction pathway controlling inhibition of DNA synthesis.

More recently, Wang et al. [ 1401 used alkaline sucrose gradient sedimentation to show that in 3.7 cells there was a strong suppression of replicon initiation 1 h after radiation which persisted for up to 6 h. In comparison, MR4 cells showed a much weaker suppression of replicon initiation and a gradual recovery 3-6 h later. No significant differences were seen in inhibition of chain elongation in the two lines. When synchronized 3.7 cells were irradiated in early S phase, they were found to have an S phase delay two to three times longer

IO

than MR cells using doses from 10 to 50 Gy. Irradiation of 3.7 cells with 10 nM staurosporine, a protein kinase inhibitor, for 3 h showed a marked reduction in the inhibition of DNA synthesis to the levels seen in MR cells. By alkaline sucrose gradient sedimentation, staurosporine was shown to reduce the inhibition of replicon initiation induced by radiation.

Much less is known about the mechanisms underlying the S phase delay than the Gi or G2 arrests. It has been pointed out [141] that in Schiz. pombe, the rad checkpoint genes may also regulate DNA replicon initiation in response to radiation since none of the rad mutants shows an S phase delay even after doses as high as 600 GY.

6.. Conclusion

Much progress has been made since the classic studies of Tolmach, Painter and others which originally described the effects of radiation on cell cycle progression. The field of altered cell cycle regulation following radiation and other genotoxic agents is now of great interest to a wide range of researchers in fields extending beyond radiobiology. Much of this interest stems from the fact that discoveries made in this area may ultimately have very practical applications, particularly in the treatment of cancers. If, in fact, following radiation or chemotherapy, cells survive by repairing DNA damage during a G2 arrest, it may be possible to increase cell kill by administering agents which force them through this block prematurely. If mutation of p53 is important in decreasing apoptosis in some tumors following radiation, this offers another potential means of manipulating radiosensitivity.

Investigations into cell cycle regulation may also help promote advances in cancer prevention by increasing our understanding of how radiation and various car- cinogens lead to malignancy. It is possible that loss of checkpoint control in non-malignant cells plays a role in the transformation process by allowing them to divide in the face of damaged DNA and accumulate chromosomal abnormalities. Understanding the mechanisms behind this could be of great importance in preventing cancers in the general population, and specifically second malignant neoplasms in patients treated with radiation and/or chemotherapy.

7. Not added in proof

Since this manuscript was submitted several recent publications have added to our understanding of how p53 leads to a Gl arrest. El-Deiry et al. (Cell 75:817-825, 1993) found that p53 results in the transcriptional activation of the WAF-l/ZIP-1 gene. Harpor et al. (Cell 75:805-816, 1993) have showed that WAF-l/CIP-1 forms complexes with the Gl cyclin-edk

A. Maity et al. /Radiother. Oncol. 31 (1994) I-13

complexes which markedly inhibits their kinase ac- tivities.

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