Growth arrest of melanoma cells is differentially regulated by contact inhibition and serum deprivation.

Both growth-factor deprivation and contact inhibition suppress cell growth; however, the mechanisms by which they inhibit cell proliferation may not be identical. The function of antiproliferative genes and the induction of programmed cell death are among the potential differences between these growth-arrest mechanisms. Specifically, an inverse relation between the expression of cyclin-dependent kinase inhibitors (CDKIs) and the susceptibility to apoptosis has been reported. To test this relation, we examined the features of growth arrest in a canine melanoma cell line, TLM1. Both contact inhibition and serum deprivation halted cell-cycle progression of TLM1 cells in the G1 phase. Prolonged growth arrest of the cells without restimulation resulted in apoptosis; conversely, the cells reentered the cell cycle after release from contact inhibition or on restimulation with serum. Cell-to-cell contact, but not serum deprivation, led to the expression of p53 and p21/Waf-1. The expression of p21/Waf-1 did not prevent apoptosis. Moreover, the ectopic overexpression of CDKIs increased apoptosis. These results support the premise that growth arrest induced by contact inhibition and serum deprivation are mediated through distinct mechanisms. Furthermore, CDKIs are not universal inhibitors of apoptosis, and in some cases, they may initiate or enhance the apoptotic program.     

p53 checkpoint-defective cells are sensitive to X rays, but not hypoxia

X-ray-induced damage leads to cell-cycle "checkpoint" arrest by p53-dependent induction of the cyclin-dependent kinase inhibitor p21 (Waf1/Cip1/Sdi1). Human tumor cells that lack this response fail to arrest after exposure to DNA-damaging agents, undergo multiple rounds of endoreduplicative DNA synthesis, and eventually commit to an apoptotic cell death. Since low oxygen tension can also induce p53 protein accumulation, and can lead to cell-cycle arrest or apoptosis, we examined the expression of p21 in tumor cells under normoxic and hypoxic conditions. In a survey of cells, mRNA for the p21 gene was induced two- to threefold in response to hypoxia in a seemingly p53-independent manner. We therefore examined genetically matched cells that differ in their p21 and p53 status for response to ionizing radiation and hypoxia. We found that both p21-deficient and p53-deficient cells exhibit an increase in chromosome instability, an increased level of apoptosis, and a failure to arrest after exposure to ionizing radiation. However, cells that lack either p21 or p53 exhibit no increase in chromosome instability or elevated apoptosis and still arrest in response to hypoxia. Thus, the mechanism responsible for the differential response to either hypoxia or X rays presumably lies in the control of cell-cycle progression in response to stress and its dependence on p21. Since the loss of a DNA-damage-dependent checkpoint does not sensitize cells to killing by stresses that elicit a DNA-damage-independent checkpoint, targeting the function of p21 pharmacologically will not kill tumor cells in situ in the absence of a DNA damage signal.     

Overexpression of CDCA2 in Human Squamous Cell Carcinoma: Correlation with Prevention of G1 Phase Arrest and Apoptosis

Cell division cycle associated 2 (CDCA2) recruits protein phosphatase 1 to chromatin to antagonize activation of ataxia telangiectasia mutated (ATM)-dependent signal transduction. ATM kinase plays a critical role in the DNA damage response and its phosphorylation cascade to inhibit the p53-MDM2 interaction, which releases p53 to induce p21 and G1 cell-cycle arrest. However, the relevance of CDCA2 to human malignancy including oral squamous cell carcinoma (OSCC) is unknown. In the current study, we found that CDCA2 expression was up-regulated in OSCC cell lines. Functional studies with shRNA system showed that knockdown of CDCA2 significantly (P<0.05) inhibited cellular proliferation compared with the control cells by arresting cell-cycle progression at the G1 phase and up-regulating the cyclin-dependent kinase inhibitors (p21^Cip1, p27^Kip1, p15^INK4B, and p16^INK4A). CDCA2 knockdown also promoted apoptosis after treatment with the DNA damage reagent, cisplatin. In clinical samples, the CDCA2 protein expression level in primary OSCCs was significantly (P<0.05) greater than in matched normal oral tissues (67/85, 79%). Furthermore, CDCA2-positive cases were correlated significantly (P<0.05) with high cancer progression. Our results showed for the first time that CDCA2 frequently is overexpressed in OSCCs and might be associated closely with OSCC progression by preventing cell-cycle arrest and apoptosis.     

The Role of Activin Type I Receptors in Activin A-Induced Growth Arrest and Apoptosis in Mouse B-Cell Hybridoma Cells

Activins transduce their signals by binding to activin type I receptors and activin type II receptors, both of which contain a serine/threonine kinase domain. In this study, we established stable transfectants expressing two types of activin receptors, ActRI and ActRIB, to clarify the role of these receptors in activin signalling for growth inhibition in HS-72 mouse B-cell hybridoma cells. Over-expression of ActRI suppressed activin A-induced cell-cycle arrest in the G1 phase caused by inhibition of retinoblastoma protein phosphorylation through induction of p21^CIP1/WAF1, a cyclin-dependent kinase inhibitor, and subsequent apoptosis. In contrast, HS-72 clones that over-expressed ActRIB significantly facilitated activin A-induced apoptosis. These results indicate that ActRI and ActRIB are distinct from each other and that the ActRI/ActRIB expression ratio could regulate cell-cycle arrest in the G1 phase and subsequent apoptosis in HS-72 cells induced by activin A.     

Inducible expression of a degradation-resistant form of p27Kip1 causes growth arrest and apoptosis in breast cancer cells

The cyclin-dependent kinase (CDK) inhibitor p27(Kip1) (p27) is an important regulator of cell cycle progression controlling the transition from G to S-phase. Low p27 levels or accelerated p27 degradation correlate with excessive cell proliferation and poor prognosis in several forms of cancer. Phosphorylation of p27 at Thr187 by cyclin E-CDK2 is required to initiate the ubiquitination-proteasomal degradation of p27. Protecting p27 from ubiquitin-mediated proteasomal degradation may increase its potential in cancer gene therapy. Here we constructed a non-phosphorylatable, proteolysis-resistant p27 mutant containing a Thr187-to-Ala substitution (T187A) which is not degraded by ubiquitin-mediated proteasome pathway, and compared its effects on cell growth, cell-cycle control, and apoptosis with those of wild-type p27. In muristerone A-inducible cell lines overexpressing wild-type or mutant p27, the p27 mutant was more resistant to proteolysis in vivo and more potent in inducing cell-cycle arrest and other growth-inhibitory effects such as apoptosis. Transduction of p27(T187A) in breast cancer cells with a doxycycline-regulated adenovirus led to greater inhibition of proliferation, more extensive apoptosis, with a markedly reduced protein levels of cyclin E and increased accumulation of cyclin D1, compared with wild-type p27. These findings support the potential effectiveness of a degradation-resistant form of p27 in breast cancer gene therapy.     

Dissociation of bone morphogenetic protein-mediated growth arrest and apoptosis of mouse B cells by HPV-16 E6/E7

We have previously found that bone morphogenetic protein-2 (BMP-2), a member of the transforming growth factor-beta family, induces cell-cycle arrest in the G1 phase and apoptotic cell death of HS-72 mouse hybridoma cells. In this study, we show that BMP-2 did not alter expression of cyclin D, cyclin E, cyclin-dependent kinase 2 (CDK2), CDK4, p27KIP1, p16INK4a, or p15INK4b, but enhanced expression of p21(CIP1/WAF1). Accumulation of p21(CIP1/WAF1) resulted in increased binding of p21(CIP1/WAF1) to CDK4 and concomitantly caused a profound decrease in the in vitro retinoblastoma protein (Rb) kinase activity of CDK4. Furthermore, the ectopic expression of human papilloma virus type-16 E7, an inhibitor of p21(CIP1/WAF1) and Rb, reverted G1 arrest induced by BMP-2. Expression of E6/E7, without increasing the p53 level, blocked inhibition of Rb phosphorylation and G1 arrest, but did not attenuate cell death in BMP-treated HS-72 cells. Taken together, these results suggest that inhibition of Rb phosphorylation by p21(CIP1/WAF1) is responsible for BMP-2-mediated G1 arrest and that BMP-2-induction of apoptosis might be independent of Rb hypophosphorylation.     

Apoptosis inhibitory activity of cytoplasmic p21(Cip1/WAF1) in monocytic differentiation

p21(Cip1/WAF1) inhibits cell-cycle progression by binding to G1 cyclin/CDK complexes and proliferating cell nuclear antigen (PCNA) through its N- and C-terminal domains, respectively. The cell-cycle inhibitory activity of p21(Cip1/WAF1) is correlated with its nuclear localization. Here, we report a novel cytoplasmic localization of p21(Cip1/WAF1) in peripheral blood monocytes (PBMs) and in U937 cells undergoing monocytic differentiation by in vitro treatment with vitamin D3 or ectopic expression of p21(Cip1/WAF1), and analyze the biological consequences of this cytoplasmic expression. U937 cells which exhibit nuclear p21(Cip1/WAF1) demonstrated G1 cell-cycle arrest and subsequently differentiated into monocytes. The latter event was associated with a cytoplasmic expression of nuclear p21(Cip1/WAF1), concomitantly with a resistance to various apoptogenic stimuli. Biochemical analysis showed that cytoplasmic p21(Cip1/WAF1) forms a complex with the apoptosis signal-regulating kinase 1 (ASK1) and inhibits stress-activated MAP kinase cascade. Expression of a deletion mutant of p21(Cip1/WAF1) lacking the nuclear localization signal (DeltaNLS-p21) did not induce cell cycle arrest nor monocytic differentiation, but led to an apoptosis-resistant phenotype, mediated by binding to and inhibition of the stress-activated ASK1 activity. Thus, cytoplasmic p21(Cip1/WAF1) itself acted as an inhibitor of apoptosis. Our findings highlight the different functional roles of p21(Cip1/WAF1), which are determined by its intracellular distribution and are dependent on the stage of differentiation.     

8-chloroadenosine induced HL-60 cell growth inhibition, differentiation, and G(0)/G(1) arrest involves attenuated cyclin D1 and telomerase and up-regulated p21(WAF1/CIP1)

8-Chloroadenosine, an active dephosphorylated metabolite of the antineoplastic agent 8-chloroadenosine 3',5'-monophosphate (8-Cl-cAMP), induces growth inhibition in multiple carcinomas. Here we report that 8-chloroadenosine inhibits growth in human promyelocytic leukemia HL-60 cells by a G(0)/G(1) phase arrest and terminates cell differentiation along the granulocytic lineage. The mechanism of 8-chloroadenosine-induced G(0)/G(1) arrest is independent of apoptosis. The expressions of cyclin D1 and c-myc in HL-60 are suppressed by 8-chloroadenosine, whereas the cyclin-dependent kinases inhibitor p21(WAF1/CIP1) is up-regulated. 8-Chloroadenosine has less effect on the expressions of cyclin-dependent kinase (cdk)2 and cdk4, G(1) phase cyclin-dependent kinases, and only moderately induces the expression of transforming growth factor beta1 (TGFbeta1) and the mitotic inhibitor p27(KIP1). Telomerase activity is reduced in extracts of 8-chloroadenosine treated HL-60 cells, but 8-chloroadenosine does not directly inhibit the catalytic activity of telomerase in vitro. Therefore, anti-proliferation of HL-60 cells by 8-chloroadenosine involves coordination of cyclin D1 suppression, reduction of telomerase activity, and up-regulation of p21(WAF1/CIP1) that arrest cell-cycle progression at G(0)/G(1) phase and terminate cell differentiation.     

Uncoupling p53 functions in radiation-induced intestinal damage via PUMA and p21

The role of p53 in tissue protection is not well understood. Loss of p53 blocks apoptosis in the intestinal crypts following irradiation but paradoxically accelerates gastrointestinal (GI) damage and death. PUMA and p21 are the major mediators of p53-dependent apoptosis and cell-cycle checkpoints, respectively. To better understand these two arms of p53 response in radiation-induced GI damage, we compared animal survival, as well as apoptosis, proliferation, cell-cycle progression, DNA damage, and regeneration in the crypts of WT, p53 knockout (KO), PUMA KO, p21 KO, and p21/PUMA double KO (DKO) mice in a whole body irradiation model. Deficiency in p53 or p21 led to shortened survival but accelerated crypt regeneration associated with massive nonapoptotic cell death. Nonapoptotic cell death is characterized by aberrant cell-cycle progression, persistent DNA damage, rampant replication stress, and genome instability. PUMA deficiency alone enhanced survival and crypt regeneration by blocking apoptosis but failed to rescue delayed nonapoptotic crypt death or shortened survival in p21 KO mice. These studies help to better understand p53 functions in tissue injury and regeneration and to potentially improve strategies to protect or mitigate intestinal damage induced by radiation.     

Correlation of STAT1 with Apoptosis and Cell-Cycle Markers in Esophageal Squamous Cell Carcinoma

We recently found evidence that STAT1 in esophageal squamous carcinoma (ESCC) cells exerts tumor suppressor function, and it regulates five key regulators of apoptosis or cell-cycle progression, including Bcl-2, Bcl-xL, survivin, cyclin D1 and p21. In this study, we confirmed these findings in four ESCC cell lines. Using immunohistochemistry, we also assessed the expression of these proteins in 62 primary tumors. The expression of these markers was heterogeneous, ranging 39 to 69% of the cohort. Significant correlation was found between STAT1 and three proteins (p21, Bcl-xL and survivin), whereas only a trend was identified for cyclin D1 and Bcl-2. We then correlated the expression of these proteins with several clinicopathologic parameters including lymph node metastasis, depth of invasion, clinical stage and overall survival. Significant correlations were found between Bcl-2 and deep invasion (p = 0.033), survivin and lymph node metastasis (p = 0.006), as well as cyclin D1 and clinical stage (p = 0.014). Patients with p21-positive tumors had a significantly longer survival compared to those with p21-negative tumors (p = 0.031). To conclude, our findings support the concept that STAT1 exerts its tumor suppressor effects in ESCC via modulating the expression of key regulators of apoptosis and cell-cycle progression.     

RASSF1A suppresses melanoma development by modulating apoptosis and cell-cycle progression

The tumor suppressor candidate gene Ras association domain family 1, isoform A (RASSF1A) encodes a microtubule-associated protein that is implicated in the regulation of cell proliferation, migration, and apoptosis. Several studies indicate that down-regulation of RASSF1A resulting from promoter hypermethylation is a frequent epigenetic abnormality in malignant melanoma. In this study, we report that compared with melanocytes in normal skins or benign skin lesions, RASSF1A is down-regulated in melanoma tissues as well as cell lines, and its expression negatively correlates with lymph node metastasis. Following ectopic expression in RASSF1A-deficient melanoma A375 cell line, RASSF1A reduces cell viability, suppresses cell-cycle progression but enhances apoptotic cell death. In vivo, RASSF1A expression inhibits the tumorigenic potential of A375 cells in nude mice, which also correlates with decreased cell proliferation and increased apoptosis. On the molecular level, ectopic RASSF1A expression leads to differential expression of 209 genes, including 26 down-regulated and 183 up-regulated ones. Among different signaling pathways, activation of the apoptosis signal-regulating kinase 1 (ASK1)/p38 MAP kinase signaling is essential for RASSF1A-induced mitochondrial apoptosis, and the inhibition of the Akt/p70S6 kinase/eIF4E signaling is also important for RASSF1A-mediated apoptosis and cell-cycle arrest. This is the first study exploring the biological functions and the underlying mechanisms of RASSF1A during melanoma development. It also identifies potential targets for further diagnosis and clinical therapy.     

MicroRNA-99a-5p suppresses breast cancer progression and cell-cycle pathway through downregulating CDC25A

In this study, we aimed to explore the association between miR-99a-5p and CDC25A in breast cancer and the regulatory mechanisms of miR-99a-5p on breast cancer. The expressions of messenger RNA and microRNAs in breast cancer tissues and adjacent tissues were analyzed by the Cancer Genome Atlas microarray analysis. Quantitative real-time polymerase chain reaction was conducted to find out the expression levels of miR-99a-5p and CDC25A. The expression levels of proteins (CDC25A, ki67, cyclin D1, p21, BAX, BCL-2, BCL-XL, MMP2, and MMP9) were determined by Western blot analysis. The relationship between miR-99a-5p and CDC25A was predicted and verified by bioinformatics analysis and dual luciferase assay. After transfection, cell proliferation, invasion, and apoptosis of breast cancer tissues were, respectively, observed by cell counting kit-8 assay, transwell assay, and flow cytometry (FCM). Furthermore, the relationship among miR-99a-5p, CDC25A, and cell-cycle progression was determined by FCM assay. The nude mouse transplantation tumor experiment was performed to verify the influence of miR-99a-5p on breast cancer cell in vivo. The expression of miR-99a-5p in breast cancer tissues and cells was significantly downregulated, whereas CDC25A expression was upregulated. MiR-99a-5p targeted CDC25A and suppressed its expression in breast cancer cells. Overexpression of miR-99a-5p and decreased expression of CDC25A could suppress breast cancer cell proliferation and invasion and facilitate apoptosis. Cell-cycle progression was significantly activated by downregulated miR-99a-5p and upregulated CDC25A. Moreover, miR-99a-5p overexpression repressed the expressions of CDC25A, marker ki67, and Cyclin D1 proteins, whereas it upregulated the expression of p21 protein. MicroRNA-99a-5p suppresses breast cancer progression and cell-cycle pathway through downregulating CDC25A.     

Myc-mediated proliferation and lymphomagenesis,but not apoptosis,are compromised by E2f1 loss

Myc and E2f1 promote cell cycle progression, but overexpression of either can trigger p53-dependent apoptosis. Mice expressing an Emu-Myc transgene in B lymphocytes develop lymphomas, the majority of which sustain mutations of either the Arf or p53 tumor suppressors. Emu-Myc transgenic mice lacking one or both E2f1 alleles exhibited a slower onset of lymphoma development associated with increased expression of the cyclin-dependent kinase inhibitor p27(Kip1) and a reduced S phase fraction in precancerous B cells. In contrast, Myc-induced apoptosis and the frequency of Arf and p53 mutations in lymphomas were unaffected by E2f1 loss. Therefore, Myc does not require E2f1 to induce Arf, p53, or apoptosis in B cells, but depends upon E2f1 to accelerate cell cycle progression and downregulate p27(Kip1).     

Retinoic acid induces CDK inhibitors and growth arrest specific (Gas) genes in neural crest cells

Retinoic acid (RA), the active metabolite of vitamin A, regulates cellular growth and differentiation during embryonic development. In excess, this vitamin is also highly teratogenic to animals and humans. The neural crest is particularly sensitive to RA, and high levels adversely affect migration, proliferation and cell death. We investigated potential gene targets of RA associated with neural crest proliferation by determining RA-mediated changes in gene expression over time, using microarrays. Statistical analysis of the top ranked RA-regulated genes identified modest changes in multiple genes previously associated with cell cycle control and proliferation including the cyclin-dependent kinase inhibitors Cdkn1a (p21), Cdkn2b (p15(INK4b)), and Gas3/PMP22. The expression of p21 and p15(INK4b) contribute to decreased proliferation by blocking cell cycle progression at G1-S. This checkpoint is pivotal to decisions regulating proliferation, apoptosis, or differentiation. We have also confirmed the overexpression of Gas3/PMP22 in RA-treated neural crests, which is associated with cytoskeletal changes and increased apoptosis. Our results suggest that increases in multiple components of diverse regulatory pathways have an overall cumulative effect on cellular decisions. This heterogeneity contributes to the pleiotropic effects of RA, specifically those affecting proliferation and cell death.