Genetic determinants of response to chemotherapy in transgenic mouse mammary and salivary tumors

Several transgenic mouse tumor models were utilized to explore how specific genetic alterations affect the tumor cell response to chemotherapeutic agents in vivo. Specifically, MMTV-ras transgenic mice were interbred to p53 knock-out mice to create a model for assessing the role of p53 in chemotherapeutic responses. In addition, MMTV-ras tumors were compared to MMTV-myc and MMTV-ras/myc tumors. Mice of each genotype reproducibly develop mammary and/or salivary tumors, but tumor growth dynamics vary considerably between genotypes. MMTV-ras/p53-/- tumors exhibit higher S phase fractions than MMTV-ras/p53+/+ tumors, although both tumor types display very low apoptosis levels. In contrast, MMTV-myc tumors exhibit both high S phase fractions and spontaneous apoptosis levels. Tumor-bearing mice of each genotype were treated with either doxorubicin or paclitaxel, and effects on overall tumor growth, cell cycle distribution and apoptosis were evaluated. Surprisingly, neither agent efficiently induced apoptosis in any of the tumor models, including those with wildtype p53. Rather, tumor responses were mediated primarily by changes in cell cycle distribution. However, the spontaneous apoptosis levels did serve as a predictor of tumor growth response, in that only those tumors with high pretreatment apoptosis levels underwent significant regression following treatment with either agent.     

Association of increased radiocurability of murine carcinomas with low constitutive expression of p21(WAF1/CIP1) protein

PURPOSE: The study investigated whether basal, constitutive levels of p21(WAF1/CIP1) protein in murine carcinomas are related to in vivo tumor radioresponse. The study is based on recent observations demonstrating that in vitro cancer cell lines are resistant to cytotoxic drugs when they express high basal levels of p21(WAF1/CIP1) protein, and that the loss of the p21 gene in the HCT116 human colorectal cancer cell line results in increased radioresponse of xenografts derived from that cell line. METHODS AND MATERIALS: Protein levels of p21(WAF1/CIP1), p53, bax, and bcl-2 were determined in 8 carcinomas (3 mammary carcinomas designated MCa-4, MCa-29, and MCa-35, 2 squamous cell carcinomas designated SCC-IV and SCC-VII, ovarian adenocarcinoma OCa-I, hepatocarcinoma HCa-I, and adenosquamous carcinoma ACa-SG) syngeneic to C3Hf/Kam mice using Western blot analysis. The tumors, growing in the right hind legs of mice, were 8 mm in diameter at the time of analysis. These tumors greatly differ in their radioresponse, assessed by TCD50 assay, and in their susceptibility to radiation-induced apoptosis. RESULTS: Protein levels of these oncogenes varied among tumors, with p21(WAF1/CIP1) showing the greatest variation: its mean densitometric value ranged from 1 to 19. Bcl-2 levels also showed broad variation in densitometric values, from 1 to 10. In comparison, bax and p53 (7 of 8 tumors contained wild-type p53) varied much less among different tumor types; their variation was within a 5-fold range, and the level of p53 was similar in 6 of 8 tumors. Tumor radioresponse correlated significantly (R = 0.77, p = 0.02) only with the magnitude of p21(WAF1/CIP1)expression: tumors with high levels of p21(WAF1/CIP1)were less radiocurable than those with lower levels. Tumor radiocurability showed a significant positive correlation (p = 0.02) with the extent of radiation-induced apoptosis, indicating that tumors that responded to radiation with higher percentages of apoptosis were more curable by radiation. Despite a strong trend to correlation, (p = 0.15), p21(WAF1/CIP1) expression did not correlate significantly with radiation-induced apoptosis, which suggested that p21(WAF1/CIP1) influenced tumor radioresponse by mechanisms beyond that of apoptosis induction. CONCLUSION: Our findings showed that murine tumors exhibit wide variation in constitutive levels of p21(WAF1/CIP1) which had a significant relationship with tumor radioresponse: tumors with high levels of p21(WAF1/CIP1) were less radiocurable than those with lower levels. These findings support the concept that p21(WAF1/CIP1) is a major determinant of tumor radioresponse in vivo, and may have important clinical implications. The pretreatment assessment of p21(WAF1/CIP1) protein could serve as a useful predictor of radiotherapy outcome and may assist in selecting an effective treatment modality.     

Mitomycin C and doxorubicin elicit conflicting signals by causing accumulation of cyclin E prior to p21WAF1/CIP1 elevation in human hepatocellular carcinoma cells

Proteins involved in the G1 phase of the cell cycle are aberrantly expressed, sometimes in mutated forms, in human cancers including human hepatocellular carcinoma. Upon attack by a DNA-damaging anticancer drug, a cell arrests at the G1 phase; this is a safety feature prohibiting entry of DNA-damaged cells into S-phase. p21WAF1/CIP1 prevents damaged cells from progressing to the next cell cycle. Here, we show that, in response to mitomycin C and doxorubicin, human hepatocellular carcinoma cells generate conflicting signals, mediated by cyclin E and p21WAF1/CIP1, which respectively accelerates and represses cell cycle transition. Exposure to these anticancer drugs led to rapid accumulation of cyclin E in both p53-proficient HepG2 and p53-deficient Hep3B cells. Such anticancer drug-induced cyclin?E accumulation influenced the G1-S-phase transition, but not DNA fragmentation-mediated death. In p53-proficient HepG2 cells, accumulation of cyclin E was followed by an increase in the level of p53-dependent p21WAF1/CIP1, thereby inhibiting further the G1-S-phase transition. Sublethal drug concentrations also induced rapid accumulation of cyclin E, but p21WAF1/CIP1 accumulation was delayed, further facilitating the G1-S-phase transition. Eventually, most cells arrested in G2/M. Thus, mitomycin C- or doxorubicin-induced conflicting signals, mediated by cyclin E and p21WAF1/CIP1, are in play in human hepatocellular carcinoma cells. Damaged G1 cells either immediately enter S-phase, or do not do so at all, depending on the extent of DNA damage.     

p21WAF1/CIP1 protein expression in primary ovarian cancer

p21WAF1/CIP1 protein is a cyclin-dependent kinase inhibitor, able to prevent the CDK2/cyclin E induced retinoblastoma protein (pRB) phosphorylation, thus inhibiting cell cycle progression at G1 phase. p21WAF1/CIP1 protein levels were examined in a series of 102 ovarian tissue samples including normal ovary, primary ovarian tumors, omental metastasis, recurrent disease and residual tumor after chemotherapy exposure, by Western blot analysis. The association of p21WAF1/CIP1 status with clinicopathological parameters and clinical outcome was also investigated. p21WAF1/CIP1 protein was detectable in 76 out of 102 (74%) ovarian tissue samples. We observed a significant trend of p21 levels to gradually increase from normal ovarian tissues (median 0 a.u.) through primary ovarian cancers (median 0.19 a.u.), omental metastases (median 0.33 a.u.) and recurrence of disease (median 0.44 a.u.) (p=0.015). In the group of stage III-IV ovarian cancer patients, p21-positive cases showed a more favourable prognosis with respect to p21-negative cases: the 3-year time to progression (TTP) rate was 58% for p21-positive compared with 33% of p21-negative cases (p=0.036). In conclusion, p21WAF1/CIP1 expression levels seem to be correlated with tumor status at the time of diagnosis and can predict TTP in a selected group of patients.     

Acteoside inhibits human promyelocytic HL-60 leukemia cell proliferation via inducing cell cycle arrest at G0/G1 phase and differentiation into monocyte

We investigated the in vitro effects of acteoside on the proliferation, cell cycle regulation and differentiation of HL-60 human promyelocytic leukemia cells. Acteoside inhibited the proliferation of HL-60 cells in a concentration- and time-dependent manner with an IC50, approximately 30 microM. DNA flow cytometric analysis indicated that acteoside blocked cell cycle progression at the G1 phase in HL-60 human promyelocytic leukemia cells. Among the G1 phase cell cycle-related proteins, the levels of cyclin-dependent protein kinase (CDK)2, CDK6, cyclin D1, cyclin D2, cyclin D3 and cyclin E were reduced by acteoside, whereas the steady-state level of CDK4 was unaffected. The protein and mRNA levels of CDK inhibitors (cyclin-dependent kinase inhibitors), such as p21(CIP1/WAF1) and p27(KIP1), were gradually increased after acteoside treatment in a time-dependent manner. In addition, acteoside markedly enhanced the binding of p21(CIP1/WAF1) and p27(KIP1) to CDK4 and CDK6, resulting in the reduction of CDK2, CDK4 and CDK6 activities. Moreover, the hypophosphorylated form of retinoblastoma increased, leading to the enhanced binding of protein retinoblastoma (pRb) and E2F1. Our results further suggest that acteoside is a potent inducer of differentiation of HL-60 cells based on biochemical activities and the expression level of CD14 cell surface antigen. In conclusion, the onset of acteoside-induced G1 arrest of HL-60 cells prior to the differentiation appears to be tightly linked to up-regulation of the p21(CIP1/WAF1) and p27(KIP1) levels and decreases in the CDK2, CDK4 and CDK6 activities. These findings, for the first time, reveal the mechanism underlying the anti-proliferative effect of acteoside on human promyelocytic HL-60 cells.     

P21-dependent g(1)arrest with downregulation of cyclin D1 and upregulation of cyclin E by the histone deacetylase inhibitor FR901228

Depsipeptide, FR901228, a novel cyclic peptide inhibitor of histone deacetylase with a unique cytotoxicity profile is currently in phase I clinical trials. Here we demonstrate that, in addition to G2/M arrest, FR901228 causes G1 arrest with Rb hypophosphorylation. In vitro kinase assays demonstrated no direct inhibition of CDK activity, however, an inhibition was observed in CDKs extracted from cells exposed to FR901228. Cyclin D1 protein disappeared between 6 and 12 hours after treatment with FR901228, whereas cyclin E was upregulated. While it did not induce wt p53, FR901228 did induce p21(WAF1/CIP1)in a p53-independent manner. Cell clones lacking p21 were not arrested in G1 phase, but continued DNA synthesis and were arrested in G2/M phase following FR901228 treatment. Finally, FR901228 blunted ERK-2/MAPK activation by EGF whereas early signal transduction events remained intact since overall cellular tyrosine phosphorylation after EGF stimulation was unaffected. Thus, FR901228, while not directly inhibiting kinase activity, causes cyclin D1 downregulation and a p53-independent p21 induction, leading to inhibition of CDK and dephosphorylation of Rb resulting in growth arrest in the early G1 phase. In contrast to the G1 arrest, the G2/M arrest is p21-independent, but is associated with significant cytotoxicity.     

Sensitivity to the non-COX inhibiting celecoxib derivative, OSU03012, is p21(WAF1/CIP1) dependent

OSU03012 is a non-COX inhibiting celecoxib derivative with growth inhibiting and apoptotic activity in many cancer cell lines. To investigate mechanisms related to cell cycle proteins in growth inhibition and apoptosis induced by OSU03012, the primary human oral epithelial cell line, TE1177, was transformed with HPV16 E6 (TE/E6), HPV16 E7 (TE/E7) or empty vector (TE/V). TE/E6 cell lines exhibiting low levels of p53 and undetectable levels of p21(WAF1/CIP1) were sensitized to the growth inhibiting and apoptotic effects of OSU03012. The TE/E7 cell lines expressing low levels of Rb and elevated levels of p53 and p21(WAF1/CIP1) were resistant. OSU03012 reduced the number of cells in the S phase of the TE/E7 and TE/V cell lines with intact p53-p21(WAF1/CIP1) checkpoint, but not in the checkpoint defective TE/E6 cell lines. Treatment with OSU03012 also markedly reduced the levels of cyclin A and Cdk2 in TE/E7 and TE/V, but not in TE/E6 cell lines, which had significantly enhanced basal levels of cyclin A and Cdk2. Consistent with the TE/E6 cell line, p21(WAF1/CIP1)-/- mouse embryo fibroblasts were more sensitive to OSU03012-induced apoptosis as evidenced by PARP and caspase 3 cleavages. These data suggest that p21(WAF1/CIP1) is an important factor in the sensitivity of cells to the growth inhibiting and apoptotic effects of OSU03012.     

The mitogen-activated protein kinase pathway mediates growth arrest or E1A-dependent apoptosis in SKBR3 human breast cancer cells

Previously, we have shown that phorbol ester (PMA) induces p21^WAF1/CIP1-dependent growth arrest in SKBr3 breast cancer and LNCaP prostate cancer cells. Here, I demonstrate that inhibition of Raf-1 kinase by dominant-negative Raf-1 or pharmacological depletion of Raf-1 prevented PMA-mediated induction of p21^WAF1/CIP1. Similarly, PD98059, a specific inhibitor of MEK, abolished p21^WAF1/CIP1induction and PMA-induced growth arrest. Like PMA, the H-ras oncogene, another activator of the Raf-1/MEK/MAPK pathway, transactivated p21^WAF1/CIP1in SKBr3 cells. I further investigated PMA-induced growth arrest following infection of SKBr3 cells with 12S E1A-expressing adenovirus. Although high levels of E1A oncoprotein prevented both PMA-induced p21^WAF1/CIP1and growth arrest, smaller amounts of E1A abrogated growth arrest without down-regulation of p21^WAF1/CIP1. Therefore, E1A can stimulate proliferation downstream of p21^WAF1/CIP1. Albeit less effective than full activity, either Rb- or p300-binding activity of E1A was sufficient for the abrogation of PMA-mediated growth arrest. E1A-driven proliferation of PMA-treated SKBr3 cells was accompanied by apoptosis. New therapeutic approaches can be envisioned that would utilize stimulation of the Raf-1/MEK/MAPK pathway to inhibit growth of PMA-sensitive cancer cells. Int. J. Cancer 78:511–517, 1998. © 1998 Wiley-Liss, Inc.     

Expression of cell-cycle regulators, cyclin E and p21WAF1/CIP1, potential prognostic markers for gastric cancer.

AIMS: The deregulation of cyclin, cyclin-dependent kinases (CDKs) and their inhibitors could have a crucial role in the development of diverse human cancers. METHODS: In this study, we analysed the expression of cyclin D1, cyclin E, p21WAF1/CIP1 and p27KIP1 in 84 surgically resected gastric cancers by immunohistochemistry with long-term follow-up (median 38 months). We also evaluated the relation between each cell cycle regulator and various clinicopathological findings, including age, sex, histological grade, tumour location, tumour type and stage and lymph-node metastasis. RESULTS: Overexpression of cyclin D1 and E was detected in 21/84 (25%) and 34/84 (40.5%) patients, respectively. Normal gastric epithelium showed consistently positive immunostain for p21WAF1/CIP1 and p27KIP1 in more than 50% of nuclei. Loss of p21WAF1/CIP1 and p27KIP1 expression was noted in 45/84 (53.6%) and 44/84 (52.4%) patients, respectively. Among the various clinicopathological findings, overexpression of cyclin E was associated with lymph-node metastasis (P=0.003) and recurrence (P=0.043). Loss of p21WAF1/CIP1 expression was more frequent in diffuse type cancers (P=0.005) and was correlated with recurrence (P=0.002) and death (P=0.002). Overexpression of cyclin E and loss of p21WAF1/CIP1 expression were significantly correlated with decreased disease-free (P=0.037; P= 0.001) and overall (P=0.031; P=0.001) survival. CONCLUSIONS: These results suggest that immunohistochemical analysis for cell cycle regulators, especially cyclin E and p21WAF1/CIP1, might be a useful prognostic indicator in gastric cancer.     

Deacetylase inhibition in malignant melanomas: impact on cell cycle regulation and survival

In the present study the deacetylase inhibitor trichostatin A (TSA) was used to elucidate the effect of protein acetylation on cell cycle progression and survival in seven human malignant melanoma cell lines. It was shown that TSA treatment led to a transient G(2)/M phase delay and accumulation of unphosphorylated retinoblastoma protein (pRB) in all cases. TSA significantly induced protein expression of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) in a dose-dependent manner in all cell lines including those not expressing p21(WAF1/CIP1) constitutively, whereas the levels of both wild-type and mutated p53 protein were reduced. The effect on p53 was not a direct result of inhibition of extracellular signal-regulated kinase-1/2 (ERK1/2) activation by TSA, as treatment of the cells with the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-1 (MEK1) inhibitor PD98059 did not result in decreased p53 protein level. Furthermore, TSA treatment led to reduction in cyclin D1 whereas cyclin D3 accumulated, the latter due to increased protein stability. Similarly, cyclin A protein was reduced whereas cyclin E level was elevated. The effect on p27(Kip1), CDK4 and CDK2 was only marginal. In all the examined cell lines, TSA treatment resulted in a profound induction of apoptosis and cleavage of poly-(ADP-ribose)-polymerase (PARP) indicative of caspase activity. Similarly, TSA-mediated apoptosis was reversed by the caspase-inhibitor z-vad-fmk. Altogether, these results suggest that p21(WAF1/CIP1) in melanomas is silenced by deacetylation, and furthermore that inhibition of deacetylation may have potential in anticancer therapy of melanoma patients.     

p21CIP-1/WAF-1 induction is required to inhibit prostate cancer growth elicited by deficient expression of the Wnt inhibitor Dickkopf-1

Osteoblastic bone metastases are the most common metastases produced by human prostate cancers (PCa). Deregulated activity of Wnt growth factors resulting from overexpression of the Wnt inhibitor Dickkopf-1 (DKK-1) is known to contribute to formation of the osteoblastic component of PCa skeletal bone metastases. In this study, we report that DKK-1 knockdown in osteolytic human PCa cells unexpectedly delays the development of both soft tissue and osseous lesions. PCa cells deficient in DKK-1 expression did not increase canonical Wnt signaling in target osteoblast cell lines; however, DKK-1 knockdown PCa cells exhibited increased expression of the CDK inhibitor p21(CIP1/WAF1) and a 32% increase in G(1) arrest compared with control cells. Ablating p21(CIP1/WAF1) in PCa cells deficient in DKK-1 was sufficient to rescue tumor growth. Collectively, our findings demonstrate that DKK-1 overexpression supports tumor growth in part by restricting expression of p21(CIP1/WAF1) through a mechanism independent of canonical Wnt signaling.     

Effects of interferon alpha on the expression of p21cip1/waf1 and cell cycle distribution in carcinoid tumors

Interferon alpha (IFN-alpha) has been shown to produce antitumor effects in 50-80% of carcinoid tumor patients and has demonstrated anti-proliferative effects in carcinoid tumor cells, but the mechanism is not well established. This study presents evidence that in a carcinoid tumor cell line, Bon1, IFN-alpha increases the expression of p21 and promotes nuclear translocation of endogenous p21. Furthermore, immunoprecipitation experiments demonstrated that p21 formed immuno-complexes with Stat1 and Stat2 in the nucleus of cells. Interferon alpha can decrease G1- and G2-phase cells, but increase S-phase population. The p21 mRNA expression is inversely correlated to the G1 population (r = -0.933, P < 0.05) and positively correlated to the S-phase population (r = 0.901, P < 0.05). In addition, IFN-alpha inhibited cyclin dependent kinases (CDK), CDK2-, CDK3-, CDK4-, and cyclin E- but not cyclin A-associated kinase activities. Immunodepletion of p21 resulted in a significant enhancement of CDK3 kinase activity (approximately 1.6-fold increase). These results suggest that the mechanism of antitumor and cell cycle regulation of IFN-alpha in carcinoid tumors may, at least in part, be p21-dependent. Based on these results, we conclude that IFN-alpha exerts antitumor effects by increased p21 expression in neuroendocrine tumors.     

The histone deacetylase inhibitor MS-275 promotes differentiation or apoptosis in human leukemia cells through a process regulated by generation of reactive oxygen species and induction of p21CIP1/WAF1 1

Effects of the histone deacetylase (HDAC) inhibitor MS-275 have been examined in human leukemia and lymphoma cells (U937, HL-60, K562, and Jurkat) as well as in primary acute myelogenous leukemia blasts in relation to differentiation and apoptosis. MS-275 displayed dose-dependent effects in each of the cell lines. When administered at a low concentration (e.g., 1 micro M), MS-275 exhibited potent antiproliferative activity, inducing p21(CIP1/WAF1)-mediated growth arrest and expression of differentiation markers (CD11b) in U937 cells. These events were accompanied by an increase in hypophosphorylated retinoblastoma protein and down-regulation of cell cycle-related proteins including cyclin D1. However, at higher concentrations (e.g., 5 micro M), MS-275 potently induced cell death, triggering apoptosis in approximately 70% of cells at 48 h. In contrast to other HDAC inhibitors such as apicidin, the extrinsic, receptor-mediated pathway played a minimal role in MS-275 lethality. However, MS-275 potently induced a very early (e.g., within 2 h) increase in reactive oxygen species (ROS), followed by the loss of mitochondrial membrane potential (Delta psi(m)) and cytosolic release of cytochrome c. These events culminated in activation of the caspase cascade, manifested by poly(ADP-ribose) polymerase, p21(CIP1/WAF1), p27(KIP), Bcl-2, and retinoblastoma protein degradation. MS-275 exposure also resulted in diminished expression of cyclin D1 and the antiapoptotic proteins Mcl-1 and XIAP. Administration of the free radical scavenger L-N-acetylcysteine blocked MS-275-mediated mitochondrial injury and apoptosis, suggesting a primary role for ROS generation in MS-275-associated lethality. Lastly, U937 cells stably expressing a p21(CIP1/WAF1) antisense construct were significantly more sensitive to MS-275-mediated apoptosis than controls, but they were impaired in their differentiation response. Together, these findings demonstrate that MS-275 exerts dose-dependent effects in human leukemia cells, i.e., p21(CIP1/WAF1)-dependent growth arrest and differentiation at low drug concentrations and a marked induction of ROS, mitochondrial damage, caspase activation, and apoptosis at higher concentrations.     

Levels of p21(WAF1/CIP1) do not affect radiation-induced cell death in human breast epithelial cells

Loss of the wild-type p53 activity and/or overexpression of the proto-oncogene bcl-2 are frequently detected in breast cancer and suggested to be related to chemotherapy and radiation therapy resistance. To identify the downstream signaling molecules for anti-proliferative and apoptotic activities of p53 and to investigate the interaction of bcl-2 with p53 in human breast epithelial cells, we have used the MCF10A cell line. We previously showed that overexpression of bcl-2 downregulates expression of p21(WAF1/CIP1) (a cyclin dependent kinase inhibitor which mediates p53 dependent G(1) arrest) and suppresses DNA damage-induced apoptosis in MCF10A cells. In the present study, we constitutively overexpressed p21(WAF1/CIP1) in bcl-2 overexpressing MCF10A cells to determine whether downregulation of p21(WAF1/CIP1) is necessary for the anti apoptotic activity of bcl-2, and to investigate the roles of p21(WAF1/CIP1) in p53-mediated cell death upon irradiation. Overexpression of p21(WAF1/CIP1) resulted in growth inhibition, but had no effect on bcl-2 inhibition of apoptosis following irradiation. Also, overexpression of p21(WAF1/CIP1) did not affect the dose-dependent radiation-induced cell lethality as determined by a clonogenic survival assay. These results suggest that bcl-2 downregulation of p21(WAF1/CIP1) is independent of the anti-apoptotic activity of bcl-2, and that p21(WAF/CIP1) is not involved in the p53-mediated cell death pathway.     

Adriamycin induced G2/M cell cycle arrest in transitional cell cancer cells with wt p53 and p21(WAF1/CIP1) genes

Adriamycin (ADM), widely used for systemic and local treatment of bladder tumors, triggers apoptosis in bladder cancer cells. Here we investigated the effect of ADM on cell cycle progression and expression of cell cycle regulating proteins in bladder cancer cell lines with various p53 and p21(WAF1/CIP1) status. Flowcytometric analysis was used to estimate the cell cycle distribution of T24, HT-1376, RT4, and SCaBER bladder cancer cell lines. Cell cycle regulating proteins were analyzed by Immunoblot. Treatment of RT4 cells, bearing wild type p53 and p21(WAF1/CIP1), with ADM induced expression of both proteins and cell cycle arrest, not in G1, as was anticipated, but in the G2 phase. Simultaneously, Retinoblastoma (Rb) protein expression was decreased. Expression of PCNA, which is a target gene of E2F, was not changed. The results suggest that even if the tumor cells bear wild type (wt) p53 and wt p21(WAF1/CIP1) and both proteins accumulate due to genotoxic stimuli, the cell cycle arrest might happen not in the G1 but in the G2 phase.