Role In Cell Cycle Checkpoints Research Articles

Abstract 1977: ACR-2316: A potentially first-in-class, potent, selective WEE1/PKMYT1 inhibitor rationally designed for superior single agent activity through synergistic disruption of cell cycle checkpoints

Abstract WEE1 and PKMYT1 kinases play essential roles in cell cycle checkpoints and DNA damage response. Genetic interaction has been documented between WEE1 and PKMYT1, and clinical trials have reported clinical activity with inhibitors of WEE1 (adavosertib, azenosertib) and PKMYT1 (lunresertib). Here, we report the discovery and characterization of ACR-2316, a dual inhibitor of WEE1 and PKMYT1 specifically designed for optimal selectivity through co-crystallography and superior single agent activity uniquely enabled by Acrivon Predictive Precision Proteomics (AP3). Mass spectrometry-based AP3 profiling was conducted across several novel WEE1 and PKMYT1 inhibitor leads generated through co-crystallography-based rational drug design. Selective leads originating from one series were further optimized using AP3 for biological structure-activity relationship analysis. AP3 profiling revealed WEE1 inhibitor-upregulated phosphorylation sites across a subset of phosphoproteins that were oppositely regulated (quenched) by PKMYT1 inhibitors. Consensus sites for these included CDK1 T14, a direct PKMYT1 phosphorylation site, as well as CHK1 S296. A particular lead compound, ACR-2316, demonstrated a desirable potent, balanced ratio of cellular WEE1 (IC50 = 2 nM, IC90 = 10 nM) and PKMYT1 (IC20 = 44 nM) inhibition resulting in superior activation of the mitotic kinases CDK1, CDK2, and PLK1 compared to adavosertib and lunresertib, based on annotated kinase substrate relationships. ACR-2316 is more selective than adavosertib, azenosertib, and lunresertib based on >200 kinases profiled by AP3 and 468 kinases assessed by KINOMEscan. Cell cycle analyses demonstrated a drastic ACR-2316-induced S-G2/M accumulation qualitatively distinct from adavosertib or lunresertib. In a 19-cancer cell line proliferation assay (CellTiter-Glo), ACR-2316 demonstrated greater potency in all cell lines tested compared to adavosertib and lunresertib (mean IC50 = 70, 252 and 364 nM, respectively). Superior anti-cancer activity of ACR-2316 was observed in 12 ovarian cancer patient-derived xenograft models tested ex vivo (CellTiter-Glo 3D) compared to azenosertib and lunresertib (mean IC50 = 9, 248 and 1620 nM, respectively). Across human tumor xenograft mouse models, oral administration of ACR-2316 demonstrated superior, durable, dose-dependent efficacy compared to azenosertib and lunresertib and was well tolerated at all doses. Complete responses observed with ACR-2316 were associated with strong WEE1 and intermediate PKMYT1 inhibition in tumors. In conclusion, ACR-2316 is a potent, selective dual WEE1/PKMYT1 inhibitor with superior single-agent activity compared to clinical WEE1 or PKMYT1 inhibitors. ACR-2316 is progressing through IND-enabling studies in preparation for clinical monotherapy development. Citation Format: Caroline Wigerup, Helén Nilsson, Lei Shi, Joon Jung, Joelle Baddour-Sousounis, Ruban Cornelius, Nina Lipjankic, Uthira Muralitharan, Valentina Siino, Ignacio Arribas Diez, Zachary Best, Martina Pasetto, William Dahlberg, Shahrzad Rafiei, Portia Lombardo, Magnus E. Jakobsson, Reina Improgo, Christina Scherer, John van Duzer, David A. Proia, Kristina Masson, Peter Blume-Jensen. ACR-2316: A potentially first-in-class, potent, selective WEE1/PKMYT1 inhibitor rationally designed for superior single agent activity through synergistic disruption of cell cycle checkpoints [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1977.

Abstract 805: A germline mutation of gene ATM p.C1899* is associated with a family history of multiple cancers in a family in Yunnan province, China

Abstract Background: Ataxia-telangiectasia mutated (ATM) is a gene that plays role in cell cycle checkpoints and DNA damage response, heterozygous germline mutations in ATM are considered as one of risk factor of breast cancer (BC). There are also some reports that ATM mutations are related to other tumor types, such as prostate cancer. Several pathogenic germline mutations in ATM have been reported included missense mutations, nonsense mutations, frameshift deletions, in-frame deletion, large deletion. Here, we report a family history of multiple cancers related to germline mutation of ATM p.C1899* in a family in Yunnan province, China. Methods: The proband is a 58-year old male patient with colon cancer. Both tumor tissue samples and peripheral blood sample of the proband was subjected to next generation sequencing (NGS) analysis by 3D Medicines Inc., Shanghai, China, a laboratory accredited by College of American Pathologists (CAP) and Clinical Laboratory Improvement Amendments (CLIA). A novel germ-line mutation was identified, that was heterozygous ATM p.C1899* mutation (c.5697C>A). Peripheral blood sample of other family members have been collected and send to 3D Medicines Inc., for NGS analysis. Results: In total of 6 members of the family underwent NGS analysis, results showed that these members were carry a germline mutation of gene ATM p.C1899*, one uncle was patient with gastric cancer, and one aunt was patient with pancreatic cancer, which means this germline mutation was related with three different cancer in a family. Conclusion: The mutation of ATM, especially germline mutation, may associated with multiple cancers, such as gastric cancer, colorectal cancer and pancreatic cancer. Accurate genetic testing should be given more attention to better control the risk of familial cancer. Citation Format: Xianghua Sun, Hushan Zhang, Wanfu Zhang, Zhiping Zhang, Bo Li. A germline mutation of gene ATM p.C1899* is associated with a family history of multiple cancers in a family in Yunnan province, China [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 805.

P53/Lactate dehydrogenase A axis negatively regulates aerobic glycolysis and tumor progression in breast cancer expressing wild-type p53.

Tumor suppressor p53 is a master regulator of apoptosis and plays key roles in cell cycle checkpoints. p53 responds to metabolic changes and alters metabolism through several mechanisms in cancer. Lactate dehydrogenase A (LDHA), a key enzyme in glycolysis, is highly expressed in a variety of tumors and catalyzes pyruvate to lactate. In the present study, we first analyzed the association and clinical significance of p53 and LDHA in breast cancer expressing wild‐type p53 (wt‐p53) and found that LDHA mRNA levels are negatively correlated with wt‐p53 but not with mutation p53 mRNA levels, and low p53 and high LDHA expression are significantly associated with poor overall survival rates. Furthermore, p53 negatively regulates LDHA expression by directly binding its promoter region. Moreover, a series of LDHA gain‐of‐function and rescore experiments were carried out in breast cancer MCF7 cells expressing endogenous wt‐p53, showing that ectopic expression of p53 decreases aerobic glycolysis, cell proliferation, migration, invasion and tumor formation of breast cancer cells and that restoration of the expression of LDHA in p53‐overexpressing cells could abolish the suppressive effect of p53 on aerobic glycolysis and other malignant phenotypes. In conclusion, our findings showed that repression of LDHA induced by wt‐p53 blocks tumor growth and invasion through downregulation of aerobic glycolysis in breast cancer, providing new insights into the mechanism by which p53 contributes to the development and progression of breast cancer.

Protective Roles of Gadd45 and MDM2 in Blueberry Anthocyanins Mediated DNA Repair of Fragmented and Non-Fragmented DNA Damage in UV-Irradiated HepG2 Cells

Growth Arrest and DNA Damage-inducible 45 (Gadd45) and MDM2 proteins, together with p21 and p53, play important roles in cell cycle checkpoints, DNA repair, and genome integrity maintenance. Gadd45 and MDM2 were activated and transcribed instantly by UV irradiation, whereas blueberry anthocyanins (BA) decreased the gene and protein expression levels in HepG2 cells for up to 24 h, and gradually restored the UV-induced fragmented and non-fragmented DNA damage of the nucleus at a time point of 12 h. Nevertheless, UV-irradiated HepG2 cell arrests occurred mainly in the G1 phase, which indicated G1 as a checkpoint. The proteins, p21 and p53, retain cellular integrity, suppressing the oncogenic transformation by interruption of the G1 phase of the cellular cycle, giving time for repairing the damage to DNA, or apoptosis induction if the damage is too severe to be repaired, while MDM2 and Gadd45 concomitantly ensure the presence of p53 and p21. Thus, we conclude that repair, together with Gadd45 and MDM2 genes, were involved in light and dark reaction mechanisms, however, BA could interfere and assist the repair through restoration, although further studies of the complex of the gene cascades triggered and responded to in BA-assisted DNA repair are needed.

An association between nuclear morphology and immunohistochemical expression of p53 and p16INK4A in lung cancer cells

Nuclear atypia is one of the most important morphological features used to diagnose malignant neoplasms. The potential molecular alteration that causes nuclear atypia remains unknown. P53 and p16INK4A play crucial roles in cell cycle checkpoints and repairing DNA damage to maintain integrity of the genome. Thus, inactivation of p53 and p16INK4A has been hypothesized to alter the chromatin structure and result in nuclear atypia. This study examined 201 primary lung cancers for the immunohistochemical expression of p53 and p16INK4A, and analyzed potential associations with the essential elements of nuclear atypia, such as nuclear size, circularity of the outline, and the density and granularity of chromatin. Tumors that expressed high levels of p53 had larger nuclei with higher chromatin density and distorted nuclear outlines. Tumors that expressed low levels of p16INK4 had larger nuclei with distorted nuclear outlines. Thus, alterations in p53 and p16INK4A may be the potential cause of nuclear atypia in neoplastic cells.

New Insights into p53 Signaling and Cancer Cell Response to DNA Damage: Implications for Cancer Therapy

Activation of the p53 signaling pathway by DNA-damaging agents was originally proposed to result either in cell cycle checkpoint activation to promote survival or in apoptotic cell death. This model provided the impetus for numerous studies focusing on the development of p53-based cancer therapies. According to recent evidence, however, most p53 wild-type human cell types respond to ionizing radiation by undergoing stress-induced premature senescence (SIPS) and not apoptosis. SIPS is a sustained growth-arrested state in which cells remain viable and secrete factors that may promote cancer growth and progression. The p21WAF1 (hereafter p21) protein has emerged as a key player in the p53 pathway. In addition to its well-studied role in cell cycle checkpoints, p21 regulates p53 and its upstream kinase (ATM), controls gene expression, suppresses apoptosis, and induces SIPS. Herein, we review these and related findings with human solid tumor-derived cell lines, report new data demonstrating dynamic behaviors of p53 and p21 in the DNA damage response, and examine the gain-of-function properties of cancer-associated p53 mutations. We point out obstacles in cancer-therapeutic strategies that are aimed at reactivating the wild-type p53 function and highlight some alternative approaches that target the apoptotic threshold in cancer cells with differing p53 status.

Abstract 4458: Chk1 inhibition as a novel therapeutic strategy for treating triple negative breast and ovarian cancers

Abstract The serine-threonine kinase Chk1 plays a critical role in cell cycle checkpoints induced in response to genotoxic stress and protecting tumour cells with a defective G1 checkpoint from DNA damage induced by cytotoxic chemotherapeutic drugs. Chk1 inhibitors are currently being pursued in the clinic as a therapeutic strategy to potentiate the anti-tumour efficacy of cytotoxic chemotherapeutic drugs without increasing their systemic toxicity. Several genetic studies have suggested that Chk1 may be critical to ensure accurate DNA replication and division during an unperturbed cell cycle and therefore Chk1 inhibitors, administered as single agents, may be beneficial anti-cancer treatments. Triple-negative breast cancer is characterised by the lack of expression of estrogen and progesterone receptors as well as Her2, along with a gene expression profile and phenotypic characteristics consistent with basal-like breast cancer. Such cancers initially respond well to cytotoxic chemotherapy and PARP inhibitors though the overall prognosis for patients with these tumour types is poor. We therefore hypothesised that triple-negative breast cancer may exhibit sensitivity to single-agent Chk1 inhibition. V158411 is a potent, selective inhibitor of the Chk1 and Chk2 kinases and potently inhibited the proliferation of triple-negative breast (average GI50 0.17µM) compared to ER-positive breast (2.3µM), lung (6.3µM) and colon (2.5µM) cancer cells. In addition, two out of three ovarian cell lines exhibited high sensitivity to growth inhibition by V158411 (SKOV-3 0.06µM and A2780 0.39µM). This sensitivity was independent of p53 mutational status, did not correlate with sensitivity to DNA damaging agents and could not be reversed in ER-positive breast cancer cells with 4-hydroxytamoxifen. Chk1 inhibition reduced triple-negative breast and ovarian cancer cell viability, activated H2AX phosphorylation and induced caspase-3/7 dependent apoptosis at doses that correlated with the anti-proliferative effects. V158411 did not induce a consistent cell cycle arrest or cells to undergo a premature mitosis. V158411 still potentiated the cytotoxicity of cisplatin and gemcitabine in p53 deficient cell lines. In an attempt to understand the sensitivity of certain cancer cell types and identify biomarkers to select sensitive patient populations, tumour cell lysates were profiled for proteins involved in the cell cycle, DNA damage response and apoptosis. Sensitivity to V158411 correlated with increased expression of pChk1(S296) and reduced expression of Mcl-1. This study suggests that triple-negative breast and ovarian cancers rely on Chk1 to complete an unperturbed cell cycle and that inhibiting Chk1 results in increased DNA damage and cell death in these tumour types. V158411, administered as a single agent or in combination with chemotherapy could be a novel, effective strategy in treating these cancer types. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4458. doi:10.1158/1538-7445.AM2011-4458

Potentiality of phosphorylation of BRCA1 at Ser 1524 to activate p21 in response to X-ray irradiation

The breast and ovarian cancer susceptibility gene BRCA1 encodes a nuclear phosphoprotein, which functions as a tumor suppressor gene. Many studies suggested that multiple functions of BRCA1 may contribute to its tumor suppressor activity, including roles in cell cycle checkpoints, apoptosis and transcription. It is postulated that phosphorylation of BRCA1 is an important means by which its cellular functions are regulated. In this study, we employed phospho-Ser-specific antibody recognizing Ser-1524 to study BRCA1 phosphorylation under conditions of DNA damage and the effects of phosphorylation on BRCA1 functions. The results showed that 10 Gy X-ray treatment significantly induced phosphorylation of Ser-1524 but not total BRCA1 protein levels. The expression both of p53 and p21 increased after irradiation, but ionizing radiation (IR) -induced activation of p21 was prior to that of p53. The percentages of G0/G1 phase remarkably increased after IR. In addition, no detectable levels of 89 kDa fragment of PARP, a marker of apoptotic cells, were observed. Data implied that IR-induced phosphorylation of BRCA1 at Ser-1524 might activate p21 protein, by which BRCA1 regulated cell cycle, but play no role in apoptosis.

AG490 influences UCN-01-induced cytotoxicity in Glioma cells in a p53-dependent fashion, correlating with effects on BAX cleavage and BAD phosphorylation

We determined the cytotoxicity of AG490 as a single agent and in combination with 7-hydroxystaurosporine (UCN-01) in a panel of malignant human glioma cell lines. Because p53 has important roles in cell cycle checkpoints, it has been anticipated that modulation of checkpoint pathways should sensitize p53 defective cells while sparing the normal cells. Cell proliferation was determined from dose–response curves. AG490 was effective as a cytotoxic agent alone regardless of p53 status. Combining the Chk1 inhibitor UCN-01 dramatically enhanced the response to AG490 in p53-mutated or deleted glioma cells. An opposite effect was noted in p53-wild type cells, in which UCN-01 and AG490 had antagonistic effects on cell proliferation and viability. We found that AG490 enhanced BAD phosphorylation in p53 wild type glioma cells, which appeared to protect against UCN-01-induced cytotoxicity, whereas AG490 enhanced UCN-01-induced cytotoxicity in p53 defective cell lines by suppression of BAD phosphorylation and induction of BAX and PARP cleavage. These observations highlight the potential for genotype-dependent factors to strongly influence response to signaling-targeted therapies in malignant gliomas and the importance of considering such factors in correlative response analyses for these agents.

RNA Silencing of Checkpoint Regulators Sensitizesp53-Defective Prostate Cancer Cells to Chemotherapy while Sparing Normal Cells

p53 is frequently mutated in patients with prostate cancer, especially in those with advanced disease. Therefore, the selective elimination of p53 mutant cells will likely have an impact in the treatment of prostate cancer. Because p53 has important roles in cell cycle checkpoints, it has been anticipated that modulation of checkpoint pathways should sensitize p53-defective cells to chemotherapy while sparing normal cells. To test this idea, we knocked down ataxia telangiectasia mutated (ATM) gene by RNA interference in prostate cancer cell lines and in normal human diploid fibroblasts IMR90. ATM knockdown in p53-defective PC3 prostate cancer cells accelerated their cell cycle transition, increased both E2F activity and proliferating cell nuclear antigen expression, and compromised cell cycle checkpoints, which are normally induced by DNA damage. Consequently, PC3 cells were sensitized to the killing effects of the DNA-damaging drug doxorubicin. Combining ATM knockdown with the Chk1 inhibitor UCN-01 further increased doxorubicin sensitivity in these cells. In contrast, the same strategy did not sensitize either IMR90 or LNCaP prostate cancer cells, both of which have normal p53. However, IMR90 and LNCaP cells became more sensitive to doxorubicin or doxorubicin plus UCN-01 when both p53 and ATM functions were suppressed. In addition, knockdown of the G(2) checkpoint regulators ATR and Chk1 also sensitized PC3 cells to doxorubicin and increased the expression of the E2F target gene PCNA. Together, our data support the concept of selective elimination of p53 mutant cells by combining DNA damage with checkpoint inhibitors and suggest a novel mechanistic insight into how such treatment may selectively kill tumor cells.

Cellular bases of the antitumor activity of a 7-substituted camptothecin in hormone-refractory human prostate carcinoma models

ST1481, a lead compound of a novel potent 7-substituted lipophilic camptothecin series, is able to overcome several mechanisms of drug resistance and was selected for clinical development. This study was designed to examine the antitumor activity of ST1481 in the treatment of preclinical models of human p53-defective hormone-refractory prostate carcinoma (DU145, PC3, and JCA-1) and to explore the cellular bases of the efficacy of camptothecins. A cellular pharmacology study (cytotoxicity, apoptosis, cellular drug accumulation, DNA damage, and cell cycle perturbation) was performed in DU145 and PC3 cells, characterized by a different cell cycle checkpoint status. The introduction of wild-type p53 in PC3 cells appreciably decreased the drug sensitivity. The 7-substituted camptothecins exhibited a high cytotoxic potency that paralleled their relative ability to induce DNA damage and a substantially increased cellular accumulation as compared to topotecan. The cytotoxic effect of camptothecins in DU145 cells was associated with arrest in S phase and early activation of apoptosis, whereas PC3 cells responded to drugs by a persistent block in G2 phase with a cytostatic effect and a late apoptosis. The efficiency of S phase checkpoint in DU145 cells was supported by a time-dependent decrease of DNA synthesis following treatment. In spite of an apparent cytostatic response and apoptosis resistance, the PC3 tumor was more responsive to in vivo treatment with camptothecins than the DU145 model. Indeed, the therapeutic outcome did not reflect the cell susceptibility to early activation of apoptosis. We suggest that cell death in PC3 cells is a delayed event consequent to persistent arrest in G2 and insufficient repair of DNA damage. ST1481 was appreciably more effective than topotecan in all tested tumors. In conclusion, the results indicated a relevant efficacy of camptothecins against human prostate carcinoma models, in spite of p53 alterations. Although p53 status could influence DNA damage and cell cycle checkpoints, p53 mutation was not a determinant of resistance. The results support that, in addition to the extent and persistence of topoisomerase I-mediated DNA damage, cell cycle checkpoints and DNA damage signaling pathways are critical determinants of tumor responsiveness to camptothecins. A role of cell cycle checkpoints activated by DNA damage in cell response is supported by the modulation of transcriptional profile.

Differential p53 Protein Expression Level in Human Cancer-Derived Cell Lines After Estradiol Treatment

Background p53 has a remarkable number of biological activities, including a central role in cell cycle checkpoints, apoptosis, senescence, and maintenance of genomic integrity. Its expression is modified by estradiol in some epithelial cancer-derived cell lines from the reproductive tract. The aim of this study was to evaluate the effect of low and high doses of estradiol in p53 gene expression in epithelial cancer-derived cell lines from the reproductive tract. Methods p53 gene expression was assessed by Northern and Western blot methods in three human epithelial cancer-derived cell lines after estradiol treatment. Results These indicated that no changes in p53 mRNA content occurred after estradiol treatment at both low (10 nM) and high (1 μM) doses of estradiol in HeLa, CaLo, and C-33 cell lines. p53 protein content was nearly constant in HeLa and C-33 cell lines at administration of 10 nM of estradiol. However, when estradiol was administered at a higher dose (1 μM), an increase in p53 protein was observed over time in HeLa and CaLo cell lines. In contrast, estradiol was without variations in C-33. Conclusions Overall results indicate that estradiol induces variations of p53 protein levels in epithelial cancer-derived cell lines from the reproductive tract in vitro and that this effect may be related with status p53 and/or presence of E6/E7 from human papillomavirus.

Uncoupling the Senescent Phenotype from Telomere Shortening in Hydrogen Peroxide-Treated Fibroblasts

Normal human cells have a limited replicative potential and inevitably reach replicative senescence in culture. Replicatively senescent cells show multiple molecular changes, some of which are related to the irreversible growth arrest in culture, whereas others resemble the changes occurring during the process of aging in vivo. Telomeres shorten as a result of cell replication and are thought to serve as a replicometer for senescence. Recent studies show that young cells can be induced to develop features of senescence prematurely by damaging agents, chromatin remodeling, and overexpression of ras or the E2F1 gene. Accelerated telomere shortening is thought to be a mechanism of premature senescence in some models. In this work, we test whether the acquisition of a senescent phenotype after mild-dose hydrogen peroxide (H2O2) exposure requires telomere shortening. Treating young HDFs with 150 μM H2O2 once or 75 μM H2O2 twice in 2 weeks causes long-term growth arrest, an enlarged morphology, activation of senescence-associated β-galactosidase, and elevated expression of collagenase and clusterin mRNAs. No significant telomere shortening was observed with H2O2 at doses ranging from 50 to 200 μM. Weekly treatment with 75 μM H2O2 also failed to induce significant telomere shortening. Failure of telomere shortening correlated with an inability to elevate p16 protein or mRNA in H2O2-treated cells. In contrast, p21 mRNA was elevated over 40-fold and remained at this level for at least 2 weeks after a pulse treatment of H2O2. The role of cell cycle checkpoints centered on p21 in premature senescence induced by H2O2 is discussed here.

Involvement of Brca1 in S-phase and G(2)-phase checkpoints after ionizing irradiation.

Cell cycle arrests in the G(1), S, and G(2) phases occur in mammalian cells after ionizing irradiation and appear to protect cells from permanent genetic damage and transformation. Though Brca1 clearly participates in cellular responses to ionizing radiation (IR), conflicting conclusions have been drawn about whether Brca1 plays a direct role in cell cycle checkpoints. Normal Nbs1 function is required for the IR-induced S-phase checkpoint, but whether Nbs1 has a definitive role in the G(2)/M checkpoint has not been established. Here we show that Atm and Brca1 are required for both the S-phase and G(2) arrests induced by ionizing irradiation while Nbs1 is required only for the S-phase arrest. We also found that mutation of serine 1423 in Brca1, a target for phosphorylation by Atm, abolished the ability of Brca1 to mediate the G(2)/M checkpoint but did not affect its S-phase function. These results clarify the checkpoint roles for each of these three gene products, demonstrate that control of cell cycle arrests must now be included among the important functions of Brca1 in cellular responses to DNA damage, and suggest that Atm phosphorylation of Brca1 is required for the G(2)/M checkpoint.

Involvement of Rb family proteins, focal adhesion proteins and protein synthesis in senescent morphogenesis induced by hydrogen peroxide.

Early passage human diploid fibroblasts develop senescent morphology prematurely within a week after a 2-hour pulse treatment with low or mild dose H(2)O(2). We test here the role of cell cycle checkpoints, cytoskeletal proteins and de novo protein synthesis in senescent morphogenesis following H(2)O(2) treatment. H(2)O(2) treatment causes transient elevation of p53 protein and prolonged inhibition of Rb hyperphosphorylation. Expression of human papillomaviral E6 gene prevented elevation of p53 but did not affect senescent morphogenesis. Expression of human papillomaviral E7 gene reduced the level of Rb protein and prevented induction of senescent morphology by H(2)O(2). The mutants of the E7 gene, in which the Rb family protein binding site was destroyed, could not reduce Rb protein or prevent H(2)O(2) from inducing senescent morphology. Senescent-like cells showed enhanced actin stress fibers. In untreated cells, vinculin and paxillin preferentially distributed along the edge of the cells. In contrast, vinculin and paxillin distributed randomly and sporadically throughout senescent-like cells. E7 expression prevented enhancement of actin filament formation and redistribution of vinculin or paxillin. Neither wild-type nor E7 cells showed changes in the protein level of actin, vinculin or paxillin measured by western blot after H(2)O(2) treatment. Finally, depletion of methionine in the culture medium after H(2)O(2) treatment prevented senescent morphogenesis without affecting dephosphorylation of Rb protein. Our results suggest that senescent morphology likely develops by a program involving activated Rb family proteins, enhancement of actin stress fibers, redistribution of focal adhesion proteins and de novo protein synthesis.