Epidermal Growth Factor Modifies Cell Cycle Control in A431 Human Squamous Carcinoma Cells Damaged by Ionizing Radiation1

[CANCER RESEARCH 54, 1407-1411. March 15. 1W] Advances in Brief

Keith R. Laderoute, Walter A. Ausserer, A. Merrill Knapp, Tandora D. Grant, and Robert M. Sutherland

Life Sciences Division, SRI /niernational, Menlo Park, California 94025 Â¡K.R. L, A. M. K., T. D. G., R. M. S.Â¡,and Dionex Corporation, Sunnyvale, California 94088 Â¡W.A. A.]

Abstract characterized in this laboratory (8). The cells were grown without antibiotics in DMEM containing 10% FBS (Gibco BRL Life Technologies, Gaithersburg, Epidermal growth factor (EGF) has been shown to radiosensitize A431 MD) in a 5% CO2 atmosphere at 37Â°C.Cultures were used within 20 passages and other human squamous carcinoma cells with high numbers of surface after recovery from frozen stocks. Culture grade murine EGF (Collaborative EGF receptors. In this study of the mechanistic basis of EGF-induced Research, Bedford, MA) was used in all EGF experiments. radiosensitization, both EGF and ionizing radiation caused Gt phase ar Irradiation Procedure. Exponentially growing A431 cells were irradiated rests in cycling A431 cells, but only radiation caused a G2-M arrest. in 60- or 100-mm plastic Petri dishes in 4 or 10 ml of medium, respectively, However, EGF enhanced the magnitude of this G2-M arrest, suggesting an by using a 137Cs7-irradiator (Mark I Model 680A; J. Shepherd and Associates, interaction of signaling pathways involved in cellular responses to EGF San Fernando, CA) at a dose rate of 250 cGy/min. All of the experiments and radiation damage. EGF and radiation also uniquely perturbed cyclin described here included untreated control cells, cells treated with EGF only, A and B, mRNA levels during the time of maximum radiation-induced cells exposed to radiation only, and cells exposed to radiation followed by the G2-M arrest. The effects of EGF on G2-M events probably originated in addition of EGF. cells in G|. It is possible that aberrant EGF signal transduction in human Cell Cycle Distribution Studies. At various times after treatment, cells squamous carcinoma cells may be exploited as a novel strategy for were trypsinized, fixed in 70% ethanol/PBS, and stored at 4Â°C.Fixed cells radiotherapy. were analyzed by flow cytometry within 2 weeks of storage. Two h before analysis, 1 X 10'' cells were resuspended in PBS containing 1 mg/ml RNase for Introduction 30 min at room temperature and then pelleted and resuspended in 10 fig/ml The contribution of eukaryotic signal transduction pathways to the propidium iodide. Propidium iodide fluorescence was measured using a biological effects of ionizing radiation is attracting much interest. Coulter Epics Elite flow cytometer (Hialeah, FL) equipped with an air-cooled Several workers have shown that ionizing radiation can induce both argon laser delivering 15 mW of light at 488 nm. The red fluorescence from 1 X IO4 cells from each sample was collected through a 610 nm bandpass filter. immediate-early and late changes in gene expression in mammalian Multicycle software (Phoenix Flow Systems, Inc., San Diego, CA) was used to cells (1, 2). Research in our laboratory has concentrated on the inter action of EGF-R2 signal transduction with the response of human SC calculate the percentage of cells in the GÃŒ,S,and G2-M phases of the cell cycle. G2 Arrest Experiments. Cell cycle distributions of A431 cells exposed to cells to ionizing radiation. EGF-R signaling in these cells has aberrant different treatments were obtained by using both asynchronous and synchro features (3) which may be exploitable therapeutically. We reported nous cultures. Exponentially growing asynchronous cells were exposed to 10 that the addition of EGF to SC cells which overexpress the EGF-R can Gy of radiation, trypsinized, and plated at densities that ensured exponential sensitize the cells to killing by ionizing radiation. EGF-induced growth at various harvest times. Immediately after plating, cells (irradiated and radiosensitization occurred when EGF was present after irradiation nonirradiated) for EGF experiments were given EGF to a concentration of 50 during the colony formation period of a clonogenic assay (4â€”6).The ng/ml (7.8 HM).Cells were harvested and processed for flow cytometry at 24, sensitization was independent of the growth inhibition commonly 48, and 72 h after plating. To synchronize A431 cultures by the double thy- observed when cell lines expressing high levels of the EGF-R are midine block method, 6.8 X IO5 cells/100-mm Petri dish (8- and 24-h time points) or 3.4 X 10s cells/100-mm Petri dish (48-h time point) were plated in treated with EGF (6). In addition, the sensitization did not involve DMEM/10% FBS containing 3 ITIMthymidine. After incubation at 37Â°Cfor 16 enhancement of DNA single strand breaks or inhibition of potentially h, the medium was replaced with DMEM/10% FBS, and the cells were incu lethal damage repair or sublethal damage repair (4). Because cell bated for another 8 h at 37Â°C.The medium was then replaced with DMEM/ cycle analysis suggested that the cell cycle distribution of A431 cells 10% FBS containing 3 HIMthymidine, and the incubation was continued for 16 may be a factor in EGF-induced radiosensitization (7), we investi h at 37Â°C.At this time, the block was released and cells were exposed to 10 Gy gated this possibility further. The findings presented here show that of radiation. Immediately after irradiation, EGF-treated cells (irradiated and EGF stimulation of the EGF-R in irradiated A431 cells enhanced the nonirradiated) were given EGF to a concentration of 50 ng/ml. All the cells classical radiation-induced G2-M arrest or checkpoint and both EGF were incubated at 37Â°Cfor 8, 24, and 48 h. At each time, the cells were washed and radiation contributed to a G, arrest. EGF and radiation also caused once with ice-cold PBS and processed for flow cytometry. unique perturbations of the levels of cyclin A and cyclin B, mRNA in GI Arrest Experiments. Exponentially growing asynchronous A431 cells A431 cells. were exposed to 10 Gy of radiation and cells (irradiated and nonirradiated) for EGF experiments were given EGF to a concentration of 50 ng/ml. Vinblastine Materials and Methods was added to a concentration of 1 fig/ml at 0, 24, and 48 h after the radiation/ EGF protocol to prevent the repopulation of G, phase by cells exiting G2 Cell Culture. The A431 human squamous carcinoma cell line was origi phase. After the addition of vinblastine, the cells were allowed to exit G! for nally obtained from the American Type Culture Collection and has been 24 h and then processed for flow cytometry. Control cells were not treated with vinblastine and were harvested at the start of the vinblastine incubation period to determine the "percentage of cells in G, at the start of vinblastine treatment". Received 12/2/93; accepted 2/4/94. Data are presented as "percentage of G! arrest," defined as The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Ihis fact. % of cells in G, after 24 h of vinblastine treatment 1This work was supported by NIH/National Cancer Institute Grants CA 37618 and CA X 100 20329. % of cells in G, at the start of vinblastine treatment 2 The abbreviations used are: EGF-R. epidermal growth factor receptor; SC, squamous carcinoma; DMEM. Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PBS, Mitotic Index Experiments. Exponentially growing A431 cells were irra phosphate-buffered saline. diated (2.5 or 10 Gy) followed immediately by the addition of EGF to a 1407

concentration of 50 ng/ml. At various times after treatment, the cells were trypsinized, swelled in 0.75 HIMKC1, fixed in methanol:acetic acid (3:1), and stained with Giemsa stain. The percentage of cells with condensed chromo somes was determined by scoring at least 3000 cells/time point. Northern Analysis. Exponentially growing A431 cells were exposed to 10 Gy of radiation followed immediately by the addition of EOF to a concentra tion of 50 ng/ml. At 8, 10, 12, and 14 h after treatment, total RNA was iso lated by using the guanidinium isothiocyanate method, purified by CsCl ul- tracentrifugation, resolved on 1% agarose/formaldehyde gels, and blotted onto nitrocellulose membranes. The blots were probed with approximately 1.6 kilobase complementary DNA fragments of human cyclins A and BI (ob tained from Dr. Tony Hunter, Salk Institute for Biological Studies, San Di ego, CA) labeled with [32P]dCTP (Dupont NEN, Boston, MA) by the random primer technique (Amersham, Arlington Heights, IL). To provide a normal ization standard for RNA loading, blots were stripped and reprobed with a DNA oligomer corresponding to a human 28S rRNA sequence (Clontech Laboratories, Palo Alto, CA) end-labeled with [32P]ATP (Amersham). 24 h 48 h 72 h

1 lUnt Â£2) EGF EGF+Rad Results EGF Enhanced the G2-M Arrest Caused by y-Irradiation of A431 Cells. A431 cells given high doses of ionizing radiation re sponded by arresting at a G2-M checkpoint, similar to the response of most eukaryotic cells subjected to substantial radiation damage. The results presented in Fig. L4 for asynchronous, exponential A431 cells exposed to 10 Gy of y-radiation show that this G2-M arrest persisted 100 for at least 72 h after irradiation. The arrest was dose dependent and 90 had a maximum value at 12 h postirradiation (data not shown). While 80 EGF exposure alone did not cause a G2-M arrest in A431 cells, EGF 70- combined with radiation enhanced the magnitude of the arrest by 48 N h posttreatment. A similar result was obtained using A431 cells syn O 60 chronized at GrS by a double thymidine block, then released from the block and given 10 Gy of radiation followed by EGF, except that the enhanced G2-M arrest caused by EGF was present at 8 h posttreatment (Fig. IB). The apparent induction of a G2-M arrest by EGF in the synchronized cell population at 24 h posttreatment is not statistically significant. EGF Prolonged a Radiation-induced Mitotic Delay in A431 Cells. A 2.5 Gy radiation dose caused a mitotic delay of 4-5 h in an asynchronous population of A431 cells (Fig. 2). EGF caused a tran 24h 48h sient delay in nonirradiated A431 cells (2-3 h) but also prolonged the I lUntE22EGFE3 RadHi EGF + Fiad delay observed in irradiated cells past 5 h. Because no more than 3% of the total population of asynchronous A431 cells were mitotic, it is Fig. 1. Histograms of results from flow cytometry showing G2-M arrests of (A} unlikely that the mitotic delay caused by EGF could be detected in the asynchronous A431 cells and (B) synchronous A431 cells, harvested at various times after treatment. Uni, untreated controls; EGF, EGF-treated; Rad, irradiated; EGF + Rad, fraction of G2-M-arrested cells determined by flow cytometry. No EGF-treated and irradiated. Bars, sample SD (n â€”3). Cells received 10 Gy of y-radiation recovery from a mitotic delay after exposure to 10 Gy of radiation and/or 50 ng/ml of EGF (7.8 DM). could be observed because of the severe morphological alterations found by 10 h posttreatment (data not shown). EGF and Radiation Caused a (., Arrest in A431 Cells. Vinblas- EGF and Radiation Had Opposing Effects on Cyclin A and B, tine was used to study the effects of EGF and radiation on the exit of mRNA Levels in A431 Cells. The radiation and EGF response of asynchronous A431 cells from Gt (9). By inhibiting formation of the cyclin A and B! mRNA expression in A431 cells are shown in Fig. 4. mitotic spindle, vinblastine prevents cells from exiting mitosis and The sampling times were chosen to span the period of maximum repopulating GÃŒ.Thus,the fraction of cells remaining in Gj can be G2-M arrest (40-45% in G2-M at 12 h) determined by flow cytometry measured as a function of time following radiation and EGF treat for asynchronous A431 cultures after exposure to radiation or radia ment. The results of these experiments are shown in Fig. 3. Note that tion combined with EGF. Consistent with literature results, the cyclin each panel in Fig. 3 applies to a different experiment; the cells were A probe hybridized more strongly with an RNA band having a lower given vinblastine at 0, 24, or 48 h posttreatment to provide different electrophoretic mobility than the band detected by the cyclin B] probe times for cell cycle effects to occur before the mitotic arrest was (10) (Fig. 4A). initiated. EGF caused a fraction of A431 cells to arrest in G] for at Separate EGF and radiation exposures had opposite effects on the least 72 h (Fig. 3), consistent with a growth-inhibitory effect of relative levels of cyclin A and B, mRNA. In the EGF-treated cells nanomolar EGF on A431 cells (9). Radiation caused a GÃŒarrestin both cyclin message levels were below the untreated control values, A431 cells by 72 h after exposure. This effect may be additive with the while the message levels in the irradiated cells ranged above those of EGF-induced GÃŒarrest. the controls by 12 h posttreatment. For example, at 12 h the normal- 1408

esized that signaling pathways associated with EGF and radiation re sponses in A431 cells interact at the level of cell cycle control. Three findings of the present study support this hypothesis: (a) EGF ampli fied a radiation-induced G2-M arrest, (b) EGF caused and radiation

Unt EGF 10 Gy EGF 4 10Gy

Hours : 10 12 14 8 10 12 14 8 IO 12 14

I Cyclin A

1345 Cyclin Bl hours Fig. 2. Mitotic index data for asynchronous A431 cells exposed to EOF (50 ng/ml; 7.8 HM),radiation (2.5 Gy) and EGF combined with radiation. Vnt, untreated controls; EGF, EGF-treated; Rad, irradiated; EGF + Rad, EGF-trealed and irradiated. The percentage of 28SrRNA cells with condensed chromosomes was determined by scoring at least 3000 cells for each data point in three sets of lOOO.Bars, sample SD (Â«= 3).

12 3456 7 8 9 IO II 12 13 14 15 16 17 18

Cyclin A

0-24 h 24-48 h 48-72 h

CD Unt I EGF I Rad I EGF + Rad 10h 12h 14 h

Fig. 3. Histograms of results from flow cytometry showing GI arrests of A431 cells in [23 EGF Rad EGF + Rad separate experiments corresponding to three different times after treatment. Unt. untreated controls; EGF, EGF-treated; Rail, irradiated; EGF + Rad, EGF-treated and irradiated. Data are presented as "percentage remaining in d," defined as Cyclin B

% of cells in G, after 24 h vinblastine treatment X 100 % of cells in G, at beginning of vinblastine treatment See the text for experimental details. Bars, sample SD (n = 3).

Â¡zedvalue of autoradiographic absorbance for cyclin A in irradiated A431 cells was 1.5 Â±0.5 (sample SD) compared with 0.4 Â±0.2 in EGF-treated cells; (n = 4; P < 0.01). Similarly, the normalized values at 12 h for cyclin B, message were 1.9 Â±0.8 and 0.6 Â±0.2 for irradiated cells and EGF-treated cells, respectively (sample standard deviations; n = 4; P < 0.01). In general, changes Â¡nthelevels of cyclin A and B, mRNA 10 to 12 h after combined radiation and EGF 8h 10h 12h 14 h exposure were distributed between the values for the radiation alone and EGF alone experiments. I EGF Rad I EGF + Rad

Fig. 4. A, typical autoradiographs from a Northern blot probed separately for human Discussion cyclin A (lop) and cyclin B, (middle) mRNA in asynchronous A431 cells harvested at 8-14 h after treatment. Arrows, positions of the 18S and 28S rRNA bands. Bottom, In A431 human SC cells, EGF can stimulate or inhibit growth, autoradiograph from the same Northern blot probed for 28S rRNA as a normalization induce differentiation, or cause cell cycle arrest (9, 11), depending standard for gel loading. B and C, histograms showing the relative values of cyclin A and cyclin BI mRNA levels from four separate experiments in autoradiographs analyzed by on its concentration in the medium (11). Flow cytometric analysis densitometry. EGF, EGF-treated; Rad, irradiated; EGF + Rad, EGF-treated and irradi has shown that EGF perturbs cell cycle progression in these cells by ated. Data are presented as ratios of autoradiographic absorbance for each pair of cyclin mRNA and 28S rRNA signals relative to the corresponding untreated control (n = 4). The causing arrests in G, and late S or G2 (9). Because radiation expo P values refer to the EGF and Rad experiments and were calculated by the Student / test sure also profoundly influences cell cycle progression, we hypoth (P < 0.01). 1409

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1994 American Association for Cancer Research. CELL CYCLE EFFECTS OF EOF IN IRRADIATED A431 CELLS enhanced a G, arrest; and (c) EOF altered the response of cyclin A noma cells "primed" with low doses (5 cGy) (17). Thus, current and B, mRNA levels to radiation. evidence shows that radiation does not invariably down-regulate cy The cell cycle distribution studies of asynchronous and synchro clin A and B, mRNA levels. It is conceivable that the observation nous A431 cultures (Fig. 1) suggest that combined EOF and radiation described here indicates the existence of feedback controls (14) that exposures influence a division delay mechanism(s), manifested as an inhibit entry into M after cyclin accumulation. enhanced G2-M arrest. Consistent with this enhancement, the combi The relevance of the present observations to the phenomenon of nation of EOF and radiation (2.5 Gy) caused a longer mitotic delay EGF-induced radiosensitization is not obvious, but a plausible hy than that resulting from radiation alone (Fig. 2). No G2-M arrest was pothesis can be formulated. Arrests in G1; S, and G2-M are well- evident in cells exposed only to EOF. Thus, the transient mitotic delay documented consequences of radiation damage, but a G2-M block is in Fig. 2 caused by EOF alone was probably a consequence of the the most common (18). Current evidence supports the position that G2- EGF-induced G] arrest shown in Fig. 3. checkpoints in eukaryotic cells delay entrance into mitosis in response EOF and radiation caused cell cycle arrests independently in A431 to unreplicated or damaged DNA, perhaps to permit restoration of cells in G, after treatment (Fig. 3). It is likely, therefore, that the genetic information (14). However, prolonged arrest in G2-M after enhanced G,-arrest observed for the combined exposure was additive exposure to a DNA-damaging agent can be cytotoxic. For example, rather than the result of a unique interaction. Nonetheless, G, A431 cisplatin treatment of murine L1210 leukemia cells (19) and exposure cells are critical targets for the investigation of EGF-induced radio- of Chinese hamster ovary cells to various cytotoxic agents (20) caused sensitization. Cells in the G, cell cycle phase contain regulatory G2-M arrests and subsequent apoptotic cell death. The enhancement molecules such as D-type cyclins that are responsive to growth factors by EGF of a G2-M arrest in irradiated A431 cells observed at 24 h like EOF (12), which has been termed a "competence" factor for the posttreatment is temporally consistent with the occurrence of EGF- G | to S-phase transition (13). Hypothetically, the enhanced G2-M induced radiosensitization, which is a late effect (4). Thus, it is pos arrest observed in irradiated A431 cells exposed to EOF was a con sible that perturbation of the recovery of irradiated A431 cells from a sequence of processes activated in G| cells. radiation-induced G2-M arrest could be cytotoxic to a subpopulation Much evidence demonstrates that the eukaryotic cell cycle is driven of cells. by families of cyclins, cyclin-dependent kinases, and their regulators In summary, both EGF and radiation caused G, arrests in cycling (12, 14). Because cyclins A and B are thought to regulate passage A431 cells, but a G2-M arrest was observed only after irradiation. through S and M phases of the cell cycle (12), we investigated the EGF and radiation perturbed cyclin A and B, mRNA levels during the effects of EGF and radiation on cyclin A and B, mRNA levels in A431 time of maximum radiation-induced G2-M arrest. EGF enhanced the cells. No significant differences in these mRNA levels were observed magnitude of the G2-M arrest at later times, suggesting an interaction in asynchronous A431 cultures at 24, 48, or 72 h posttreatment (data of signaling pathways involved in cellular responses to EGF and not shown). However, substantial differences in the relative levels of radiation damage. These results pose two basic questions for further cyclin A and B, messages were observed after irradiation during the research: what is the role of G! regulators in this interaction in A431 period of maximum G2-M arrest (8-14 h; Fig. 4). Analysis of these cells; and does the EGF enhancement of a G2-M arrest contribute to results suggests that at early times different mechanisms determined cell death? Further research should determine whether aberrant signal the effects of radiation and EGF on cyclin A and B, messages in these transduction in human SC cells like A431 can be exploited as a novel cells. EGF suppressed the accumulation of message levels relative to strategy for radiotherapy. those of the untreated cells. This response is consistent with the Acknowledgments mitotic delay observed at short times in EGF-treated cells (Fig. 2) and may be related to the ability of the growth factor to arrest a proportion We thank T. Hunter for providing cDNA probes for human cyclins A and BI. of G! cells in the asynchronous population (Fig. 3). Compared with the EGF effect, radiation did not suppress the accumulation of cyclin References A and B, mRNA levels and caused an increase in these levels relative 1. Weichselbaum, R. R., Hallaban, D. E., Sukhatme, V., Dritschilo, A., Sherman, M. D., to the asynchronous control cells by 12 h posttreatment. Cyclin A and and Kufe, D. W. Biological consequences of gene regulation after ionizing radiation exposure. J. Nati. Cancer Inst., 83: 480-484, 1991. B, mRNA levels normally increase as synchronized cells progress 2. Fuks, Z., and Weichselbaum, R. R. Radiation tolerance and the new biology: growth through G2 (10). Given this previous observation, the accumulation of factor involvement in radiation injury to the lung. Int. J. RadiÃ¢t.Oncol. Biol. Phys., cyclin messages detected in the irradiated A431 cells is consistent with 24: 183-184, 1992. 3. Mansbridge, J. M., Knuchel, R., Knapp, A. M., and Sutherland, R. M. Importance of the radiation-induced synchronization of the cells at a G2-M arrest. In tyrosine phosphatases in the effects of cell-cell contact and microenvironments on view of the opposite effects of EGF and radiation on cyclin A and B, EGF-stimulated tyrosine phosphorylation. J. Cell. Physiol., 151: 433-442, 1992. message accumulation, changes in the levels of these messages in 4. Kwok, T. T., and Sutherland, R. M. Enhancement of sensitivity of human squamous carcinoma cells to radiation by epidermal growth factor. J. Nati. Cancer Inst., 81: irradiated A431 cells exposed to EGF probably represent the combi 1020-1024, 1989. nation of the separate effects of these two agents. Whether the per 5. Kwok, T. T., and Sutherland, R. M. Epidermal growth factor modification of radiore- sistance related to cell-cell interactions. Int. J. RadiÃ¢t.Oncol. Biol. Phys., 20: 315- turbations of these message levels detected at early times have a 318, 1991. causal role in the EGF enhancement of the G2-M arrest observed 6. Kwok, T. T., and Sutherland, R. M. Differences in EGF related radiosensitization of between 24 and 48 h posttreatment of asynchronous cells is not known human squamous carcinoma cells with high and low numbers of EGF receptors. Br. J. Cancer, 64: 251-254, 1991. presently. 7. Kwok, T. T., and Sutherland, R. M. Cell cycle dependence of epidermal growth factor The influence of ionizing radiation on the expression of genes for induced radiosensitization. Int. J. RadiÃ¢t.Oncol. Biol. Phys., 22.- 525-527, 1992. cyclins and other cell cycle control proteins appears to depend on cell 8. Knuechel, R., Keng, P., Hofstaedter, F., Langmuir, V., Sutherland, R. M., and Penney, D. P. Differentiation patterns in two- and three-dimensional culture systems of human type and position in the cell cycle. The accumulation of cyclin B squamous carcinoma cell lines. Am. J. Pathol., 737: 725-736, 1990. mRNA normally associated with progression through G2 was inhib 9. MacLeod, C. L., Luk, A., Castagnola, J., Cronin, M., and Mendelsohn, J. EGF induces ited by X-rays in HeLa cells, suggesting a mechanism of G2-M arrest cell cycle arrest of A431 human epidermoid carcinoma cells. J. Cell. Physiol., 727: 175-182, 1986. (15). Transient depression of cyclin A and B mRNA was also de 10. Pines, J., and Hunter, T. Human cyclin A is adenovirus ElA-associated protein p60 scribed for U-937 and HL-60 leukemia cells treated with high doses and behaves differently from cyclin B. 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13. Aaronson, S. A. Growth factors and cancer. Science (Washington DC), 254: 1146- adaptive survival responses. In: T. Sugahara, L. A. Sagan, and T. Aoyama (eds.). Low 1153, 1991. Dose Irradiation and Biological Defense Mechanisms, pp. 263-266. Amsterdam: 14. Murray, A. W. Creative blocks: cell-cycle checkpoints and feedback controls. Nature Elsevier Science Publishers, 1992. (Lond.), 359: 599-604, 1992. 18. Weinert, T., and Lydall, D. Cell cycle checkpoints, genetic instability and cancer. 15. Muschel, R. J., Zhang, H. B., lliakis, G., and McKenna, W. G. Effects of ionizing Semin. Cancer Biol., 4: 129-140, 1993. radiation on cyclin expression in HeLa cells. RadiÃ¢t.Res., 132: 153-157, 1992. 19. Sorenson, C. M., Barry, M. A., and Eastman, A. Analysis of events associated with 16. Dalla, R., Mass, R., Gunjii. H., Weichselbaum, R., and Kufe, D. Down-regulation of cell cycle arrest at G2 phase and cell death induced by cisplalin. J. Nati. Cancer Inst., cell cycle control genes by ionizing radiation. Cell Growth & Differ., 3: 637-644, 82: 749-755, 1990. 1992. 20. Barry, M. A., Behnke, C. A., and Eastman, A. Activation of programmed cell death 17. Meyers, M., Schea, R. A., Petrowski, A. E., Seabury, H., McLaughlin, P. W., Lee, I., (apoptosis) by cisplatin, other anticancer drugs, toxins and hyperthermia. Biochem. Lee, S. W., and Boothman, D. A. Role of X-ray-inducible genes and proteins in Pharmacol., 40: 2353-2362, 1990.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1994 American Association for Cancer Research. Epidermal Growth Factor Modifies Cell Cycle Control in A431 Human Squamous Carcinoma Cells Damaged by Ionizing Radiation

Keith R. Laderoute, Walter A. Ausserer, A. Merrill Knapp, et al.

Cancer Res 1994;54:1407-1411.

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