P53-defective Cell Research Articles

Downregulation of BMI-1 By the Small Molecule PTC596 Induces Mitochondrial Apoptosis in Mantle Cell Lymphoma

▪Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin lymphoma typically associated with the t(11;14) translocation, resulting in overexpression of cyclin D1 (CCND1). Although the clinical approval of the first-in-class Bruton's tyrosine kinase (BTK) inhibitor ibrutinib provides a new treatment option, the great majority of patients with MCL remain incurable. Studies are needed to optimize the role of ibrutinib in MCL therapy and to address ibrutinib resistance. BMI-1 is crucially involved in cancer stem cell self-renewal and maintenance. BMI-1 has been shown to be up-regulated in MCL cells, especially in tumor-initiating side population (SP) cells (Teshima et al. Oncogene 2014). Importantly, BMI-1 levels are higher in refractory/relapsed patients than those at initial diagnosis. PTC-209 and PTC596 are recently developed novel small-molecule selective BMI-1 inhibitors with different modes of action (Kreso et al. Nat Med 2014). PTC-209 inhibits BMI-1 production while PTC596 accelerates BMI-1 degradation. We have reported anti-leukemia effects of the BMI-1 inhibitors PTC-209 and PTC596 (Cancer Sci 2015; Blood Cancer J 2017) in acute myeloid leukemia cell lines and patient-derived leukemia blasts. In this study, we investigated the effects of PTC-209 and PTC596 in MCL. Oncomine analysis showed that BMI1 mRNA levels were higher in MCL cells than in normal B cells. The levels were not specifically associated with CCND1 levels, ATM, CDKN2A or TP53 mutations, or patient prognosis, implying that BMI1 overexpression may be a fundamental abnormality in MCL. PTC-209 and PTC596 inhibited cell growth and induced apoptosis in 8 MCL cell lines in a dose- and time-dependent manner. Their IC50 values (concentration at which cell growth is inhibited by 50%) ranged from 1.5 to 11.2 µM for PTC-209 and from 68 to 340 nM for PTC596. ED50 values (effective concentration inducing 50% cell killing as measured by annexin V positivity) ranged from 2.7 to > 50 µM for PTC-209 and from 150 to 507 nM for PTC596. PTC596 was > 10 times more potent than PTC-209. IC50 and ED50 values of PTC-209 significantly correlated with those of PTC596 [r =0.94 (P= 0.0004) for IC50 and r =0.85 (P= 0.015) for ED50], supporting the idea that their anti-lymphoma activities depend on downregulation of BMI-1. In support of this, high BMI-1 protein levels predicted high susceptibility to PTC596 (r = -0.88; P=0.0039). The anti-lymphoma effects of PTC596 appeared to be p53-independent since IC50 and ED50 values were not significantly different against p53 wild-type and p53 defective cell lines. In addition, stable knockdown of p53 did not significantly affect MCL cell susceptibility to PTC596. Activity of PTC596 was independent of MCL cell susceptibility to ibrutinib. PTC596 induced mitochondrial apoptosis, as evidenced by loss of mitochondrial membrane potential, caspase-3 cleavage, BAX activation and phosphatidylserine externalization in MCL cells. PTC596 effectively decreased SP cells (by 97.3% at 150 nM, 99.8% at 300 nM and >99.9% at 600 nM PTC596) compared to ibrutinib that decreased SP cells by 33% even at 5 µM ibrutinib. Combination of PTC596 with bortezomib or doxorubicin showed additive anti-proliferative and apoptotic effects. The averaged combination index values were approximately 1, ranging from 0.95 to 1.18. Interestingly, PTC596 synergized with ibrutinib in inducing cell death, through modulation of the BCL-2 family protein MCL-1. Collectively, BMI-1 inhibition may offer a novel therapeutic strategy for MCL. DisclosuresLiangl:PTC Therapeutics: Employment. Weetall:PTC Therapeutics: Employment. Branstrom:PTC Therapeutics: Employment.

The antiproliferative effects of ataxia-telangiectasia mutated and ATM- and Rad3-related inhibitions and their enhancements with the cytotoxicity of DNA damaging agents in cholangiocarcinoma cells.

To investigate whether the inhibitions of ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) kinases by their specific inhibitors, KU-55933 and VE-821, respectively, are able to promote the cytotoxic activity of genotoxic agents including gemcitabine, 5-Fluorouracil, cisplatin and doxorubicin, in cholangiocarcinoma (CCA) and immortalized cholangiocyte cell lines. Cell viability of cells treated with DNA damaging agents, alone and in combination with KU-55933 and VE-821, was determined by MTT assay. The changes of cell cycle distribution were evaluated by flow cytometry analysis. Colony formation was conducted to assess the effects of KU-55933 and VE-821 on cell proliferation. The levels of protein expression and phosphorylation were examined by western blot analysis. The cytotoxic effects of DNA damaging agents varied among CCA cell lines. Each DNA damaging drug induced different phases of the cell cycle in CCA cells. The combinations of both KU-55933 and VE-821 with DNA damaging agents promoted more cytotoxic activity than single inhibition in some CCA cell lines. ATM and ATR inhibitors decreased the effects of DNA damaging agent-induced ATM-Chk2 and ATR-Chk1 activations in CCA cells. Inhibitions of ATM and ATR potentiated the cytotoxic effects of DNA damaging agents in CCA cells, especially p53 defective HuCCA1 and RMCC1 cell lines.

Mutant TP53 G245C and R273H promote cellular malignancy in esophageal squamous cell carcinoma

BackgroundTP53 gene mutations occur in more than 50% of human cancers and the vast majority of these mutations in human cancers are missense mutations, which broadly occur in DNA binding domain (DBD) (Amino acids 102–292) and mainly reside in six “hotspot” residues. TP53 G245C and R273H point mutations are two of the most frequent mutations in tumors and have been verified in several different cancers. In the previous study of the whole genome sequencing (WGS), we found some mutations of TP53 DBD in esophageal squamous cell carcinoma (ESCC) clinical samples. We focused on two high-frequent mutations TP53 p.G245C and TP53 p.R273H and investigated their oncogenic roles in ESCC cell lines, p53-defective cell lines H1299 and HCT116 p53−/−.ResultsMTS and colony formation assays showed that mutant TP53 G245C and R273H increased cell vitality and proliferation. Flow cytometry results revealed inhibition of ultraviolet radiation (UV)- and ionizing radiation (IR)- induced apoptosis and disruption of TP53-mediated cell cycle arrest after UV, IR and Nocodazole treatment. Transwell assays indicated that mutant TP53 G245C and R273H enhanced cell migration and invasion abilities. Moreover, western blot revealed that they were able to suppress the expression of TP53 downstream genes in the process of apoptosis and cell cycle arrest induced by UV, which suggests that these two mutations can influence apoptosis and growth arrest might be due, at least in part, to down-regulate the expression of P21, GADD45α and PARP.ConclusionsThese results indicate that mutant TP53 G245C and R273H can lead to more aggressive phenotypes and enhance cancer cell malignancy, which further uncover TP53 function in carcinogenesis and might be useful in clinical diagnosis and therapy of TP53 mutant cancers.

Etoposide radiosensitizes p53-defective cholangiocarcinoma cell lines independent of their G2 checkpoint efficacies.

Radiotherapy has been accounted as the most comprehensive cancer treatment modality over the past few decades. However, failure of this treatment modality occurs in several malignancies due to the resistance of cancer cells to radiation. It was previously reported by the present authors that defective cell cycle checkpoints could be used as biomarkers for predicting the responsiveness to radiation in individual patients with cholangiocarcinoma (CCA). However, identification of functional defective cell cycle checkpoints from cells from a patient's tissues is cumbersome and not applicable in the clinic. The present study evaluated the radiosensitization potential of etoposide in p53-defective CCA KKU-M055 and KKU-M214 cell lines. Treatment with etoposide enhanced the responsiveness of two p53-defective CCA cell lines to radiation independent of G2 checkpoint function. In addition, etoposide treatment increased radiation-induced cell death without altering the dominant mode of cell death of the two cell lines. These findings indicate that etoposide could be used as a radiation sensitizer for p53-defective tumors, independent of the function of G2 checkpoint.

Chk1 inhibition significantly potentiates activity of nucleoside analogs in TP53-mutated B-lymphoid cells.

Treatment options for TP53-mutated lymphoid tumors are very limited. In experimental models, TP53-mutated lymphomas were sensitive to direct inhibition of checkpoint kinase 1 (Chk1), a pivotal regulator of replication. We initially tested the potential of the highly specific Chk1 inhibitor SCH900776 to synergize with nucleoside analogs (NAs) fludarabine, cytarabine and gemcitabine in cell lines derived from B-cell malignancies. In p53-proficient NALM-6 cells, SCH900776 added to NAs enhanced signaling towards Chk1 (pSer317/pSer345), effectively blocked Chk1 activation (Ser296 autophosphorylation), increased replication stress (p53 and γ-H2AX accumulation) and temporarily potentiated apoptosis. In p53-defective MEC-1 cell line representing adverse chronic lymphocytic leukemia (CLL), Chk1 inhibition together with NAs led to enhanced and sustained replication stress and significantly potentiated apoptosis. Altogether, among 17 tested cell lines SCH900776 sensitized four of them to all three NAs. Focusing further on MEC-1 and co-treatment of SCH900776 with fludarabine, we disclosed chromosome pulverization in cells undergoing aberrant mitoses. SCH900776 also increased the effect of fludarabine in a proportion of primary CLL samples treated with pro-proliferative stimuli, including those with TP53 disruption. Finally, we observed a fludarabine potentiation by SCH900776 in a T-cell leukemia 1 (TCL1)-driven mouse model of CLL. Collectively, we have substantiated the significant potential of Chk1 inhibition in B-lymphoid cells.

KAVA Chalcone, Flavokawain A, Inhibits Urothelial Tumorigenesis in the UPII-SV40T Transgenic Mouse Model

Flavokawain A (FKA) is the predominant chalcone identified from the kava plant. We have previously shown that FKA preferentially inhibits the growth of p53 defective bladder cancer cell lines. Here, we examined whether FKA could inhibit bladder cancer development and progression in vivo in the UPII-SV40T transgenic model that resembles human urothelial cell carcinoma (UCC) with defects in the p53 and the retinoblastoma (Rb) protein pathways. Genotyped UPII-SV40T mice were fed orally with vehicle control (AIN-93M) or FKA (6 g/kg food; 0.6%) for 318 days starting at 28 days of age. More than 64% of the male mice fed with FKA-containing food survived beyond 318 days of age, whereas only about 38% of the male mice fed with vehicle control food survived to that age (P = 0.0383). The mean bladder weights of surviving male transgenic mice with the control diet versus the FKA diet were 234.6 ± 72.5 versus 96.1 ± 69.4 mg (P = 0.0002). FKA was excreted primarily through the urinary tract and concentrated in the urine up to 8.4 μmol/L, averaging about 38 times (males) and 15 times (females) more concentrated than in the plasma (P = 0.0001). FKA treatment inhibited the occurrence of high-grade papillary UCC, a precursor to invasive urothelial cancer, by 42.1%. A decreased expression of Ki67, survivin, and X-linked inhibitor of apoptotic proteins (XIAP) and increased expression of p27 and DR5, and the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive apoptotic cells were observed in the urothelial tissue of FKA-fed mice. These results suggest a potential of FKA in preventing the recurrence and progression of non-muscle-invasive UCC.

PARG dysfunction enhances DNA double strand break formation in S-phase after alkylation DNA damage and augments different cell death pathways

Poly(ADP-ribose) glycohydrolase (PARG) is the primary enzyme responsible for the degradation of poly(ADP-ribose). PARG dysfunction sensitizes cells to alkylating agents and induces cell death; however, the details of this effect have not been fully elucidated. Here, we investigated the mechanism by which PARG deficiency leads to cell death in different cell types using methylmethanesulfonate (MMS), an alkylating agent, and Parg−/− mouse ES cells and human cancer cell lines. Parg−/− mouse ES cells showed increased levels of γ-H2AX, a marker of DNA double strand breaks (DSBs), accumulation of poly(ADP-ribose), p53 network activation, and S-phase arrest. Early apoptosis was enhanced in Parg−/− mouse ES cells. Parg−/− ES cells predominantly underwent caspase-dependent apoptosis. PARG was then knocked down in a p53-defective cell line, MIAPaCa2 cells, a human pancreatic cancer cell line. MIAPaCa2 cells were sensitized to MMS by PARG knockdown. Enhanced necrotic cell death was induced in MIAPaCa2 cells after augmenting γ-H2AX levels and S-phase arrest. Taken together, these data suggest that DSB repair defect causing S-phase arrest, but p53 status was not important for sensitization to alkylation DNA damage by PARG dysfunction, whereas the cell death pathway is dependent on the cell type. This study demonstrates that functional inhibition of PARG may be useful for sensitizing at least particular cancer cells to alkylating agents.

Molecular mechanism implicated in Pemetrexed-induced apoptosis in human melanoma cells

BackgroundMetastatic melanoma is a lethal skin cancer and its incidence is rising every year. It represents a challenge for oncologist, as the current treatment options are non-curative in the majority of cases; therefore, the effort to find and/or develop novel compounds is mandatory. Pemetrexed (Alimta®, MTA) is a multitarget antifolate that inhibits folate-dependent enzymes: thymidylate synthase, dihydrofolate reductase and glycinamide ribonucleotide formyltransferase, required for de novo synthesis of nucleotides for DNA replication. It is currently used in the treatment of mesothelioma and non-small cell lung cancer (NSCLC), and has shown clinical activity in other tumors such as breast, colorectal, bladder, cervical, gastric and pancreatic cancer. However, its effect in human melanoma has not been studied yet.ResultsIn the current work we studied the effect of MTA on four human melanoma cell lines A375, Hs294T, HT144 and MeWo and in two NSCLC cell lines H1299 and Calu-3. We have found that MTA induces DNA damage, S-phase cell cycle arrest, and caspase- dependent and –independent apoptosis. We show that an increment of the intracellular reactive oxygen species (ROS) and p53 is required for MTA-induced cytotoxicity by utilizing N-Acetyl-L-Cysteine (NAC) to blockage of ROS and p53-defective H1299 NSCLC cell line. Pretreatment of melanoma cells with NAC significantly decreased the DNA damage, p53 up-regulation and cytotoxic effect of MTA. MTA was able to induce p53 expression leading to up-regulation of p53-dependent genes Mcl-1 and PIDD, followed by a postranscriptional regulation of Mcl-1 improving apoptosis.ConclusionsWe found that MTA induced DNA damage and mitochondrial-mediated apoptosis in human melanoma cells in vitro and that the associated apoptosis was both caspase-dependent and –independent and p53-mediated. Our data suggest that MTA may be of therapeutic relevance for the future treatment of human malignant melanoma.

Design and synthesis of 2-heterocyclyl-3-arylthio-1H-indoles as potent tubulin polymerization and cell growth inhibitors with improved metabolic stability.

New arylthioindoles (ATIs) were obtained by replacing the 2-alkoxycarbonyl group with a bioisosteric 5-membered heterocycle nucleus. The new ATIs 5, 8, and 10 inhibited tubulin polymerization, reduced cell growth of a panel of human transformed cell lines, and showed higher metabolic stability than the reference ester 3. These compounds induced mitotic arrest and apoptosis at a similar level as combretastatin A-4 and vinblastine and triggered caspase-3 expression in a significant fraction of cells in both p53-proficient and p53-defective cell lines. Importantly, ATIs 5, 8, and 10 were more effective than vinorelbine, vinblastine, and paclitaxel as growth inhibitors of the P-glycoprotein-overexpressing cell line NCI/ADR-RES. Compound 5 was shown to have medium metabolic stability in both human and mouse liver microsomes, in contrast to the rapidly degraded reference ester 3, and a pharmacokinetic profile in the mouse characterized by a low systemic clearance and excellent oral bioavailability.

Knockdown of CITED2 using short‐hairpin RNA sensitizes cancer cells to cisplatin through stabilization of p53 and enhancement of p53‐dependent apoptosis

CITED2 is a transcriptional modulator which has been implicated in human oncogenesis. In the present study, we examined whether CITED2 is also involved in the resistance of cancer cells to the chemotherapeutic drug cisplatin. We first observed that knockdown of CITED2 using short-hairpin RNA sensitized non-tumorigenic HEK293 cells to cisplatin. Sensitization to cisplatin following knockdown of CITED2 was also observed in cervical carcinoma HeLa cells and in cisplatin-resistant HeLa cells, thereby showing that acquired cisplatin resistance could be reversed by CITED2 knockdown. This sensitization response was dependent on the status of p53 since efficient sensitization was observed in p53-positive hepatocellular carcinoma (HCC) Sk-Hep-1 cells, whereas a negligible response was produced in the two p53-defective cell lines HCC Mahlavu and lung cancer H1299. In contrast, overexpression of CITED2 decreased sensitivity of HEK293 cells to cisplatin, while moderate resistance was produced in HeLa cells. Overexpression of CITED2 also decreased sensitivity to cisplatin in p53-defective H1299 cells when exogenous p53 expression was re-introduced. We observed that knockdown of CITED2-induced CBP/p300-mediated p53 acetylation (Lys373) in HEK293 cells, thereby leading to a decrease of p53 ubiquitination and subsequent accumulation of the p53 protein. Notably, the effects of CITED2 knockdown on p53 accumulation and the increase of p53's target Bax were more pronounced after treatment with cisplatin. Based on these results, we propose that a combination of cisplatin and CITED2 shRNA may represent an effective treatment against p53-sensitive cancer cells.

Abstract 1759: MK-1775, a novel Wee1 kinase inhibitor, radiosensitizes p53-defective non-small cell lung cancer (NSCLC) cells

Abstract Wee1 is a tyrosine kinase involved in the S>G2 and G2>M cell cycle transitions. Following DNA damage, Wee1 is phosphorylated by upstream kinases and stabilized. Stabilized Wee1, in turn, phosphorylates and inhibits CDC2, preventing the cells from entering mitosis before repairing their damaged DNA. Abrogation of Wee1 activity, either by decreasing expression using siRNA, or by inhibiting protein activity with selective small molecule inhibitors, sensitizes tumor cells to DNA-damaging agents. This sensitization preferentially takes place in a p53 null or mutant context. We have investigated the ability of a novel, small molecule inhibitor of Wee1, MK-1775, to radiosensitize NSCLC cells treated in vitro. Four NSCLC cell lines were used in the study: A549 and H460 with wild-type p53 status and H1299 and Calu6 with null or mutant p53 status, respectively. A 24 hr pre-irradiation treatment with 200 nM MK-1775 suppressed phosphorylation of CDC2 in both A549 and H1299 cells, however, this treatment did not produce a radiosensitizing effect in either line. An 18 hr post-irradiation treatment slightly radiosensitized the H1299 line but not the A549 line. The optimal treatment of a 1 hr pre-irradiation followed by an 18 hr post-irradiation treatment substantially radiosensitized the H1299 and Calu6 lines, yielding dose-enhancement factors (DEFs) at 10% survival of 1.39 and 1.47 respectively, but did not sensitize the A549 or H460 cells, DEFs of 1.0. To determine the mechanism responsible for the radiosensitizing effect of MK-1775, cells were treated with nocodazole following a dose of 4 Gy and analyzed for phospho-histone H3 as an indication of mitotic index after 4 hrs. Unirradiated cells accumulated in mitosis during the 4 hrs in nocodazole and this accumulation was substantially enhanced by MK-1775 in both p53 wild type A549 and p53 null H1299 cells suggesting that Wee1 inhibition accelerates unirradiated cells into mitosis prematurely. Neither A549 nor H1299 cells, irradiated with 4 Gy, accumulated in mitosis during the 4 hr nocodazole treatment in the absence of MK-1775 indicating a robust G2 block for both lines. However, H1299 cells irradiated with 4 Gy and treated with MK-1775 accumulated in mitosis to a level higher than the unirradiated cells treated with MK-1775 alone. This enhanced entry of irradiated cells into mitosis was not evident to nearly the same degree in the A549 cells. Thus, we conclude that the Wee1 inhibitor, MK-1775, accelerates cells harboring DNA-damage prematurely into mitosis in a p53-dependent manner correlating with its radiosensitizing effects. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1759.

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.

Radiosensitization of lung cancer by nutlin, an inhibitor of murine double minute 2

p53 plays a critical role in cell cycle arrest and induction of apoptosis. Certain malignancies carry wild-type p53, which is frequently down-regulated by murine double minute 2 (MDM2) overexpression. Availability of a small-molecule inhibitor against MDM2, nutlin, has made it feasible to evaluate the anti-MDM2-based therapeutic strategies. The rationale for the current study is that functional p53 has been linked with improved responses to radiation treatment. Hence, this study evaluates the use of nutlin, a small-molecule inhibitor that blocks the interaction of p53 and MDM2, in sensitizing cancer cells to radiation. Expression of MDM2, p53, and p21 in both p53 wild-type and p53-defective lung cancer cell lines was examined. Clonogenic and 7-amino-actinomycin D studies were used to determine possible mechanisms of cell death. The combined effect of MDM2 inhibition and radiation on cell cycle was also studied. We found that radiosensitization by nutlin occurs in lung cancer cells with wild-type p53. There were increased apoptosis and cell cycle arrest following administration of nutlin and radiation. Furthermore, the combination of nutlin and radiation decreased the ability of endothelial cells to form vasculature, as shown by Matrigel assays. Our data suggest that nutlin is an effective radiosensitizer of p53 wild-type cells. The radiosensitizing effect seems to be at least partially due to induction of apoptosis and cell cycle arrest. In addition, nutlin may be an effective radiosensitizer of tumor vasculature.

P53-Defective Tumors With a Functional Apoptosome-Mediated Pathway: A New Therapeutic Target

Although cancer cells appear to maintain the machinery for intrinsic apoptosis, defects in the pathway develop during malignant transformation, preventing apoptosis from occurring. How to specifically induce apoptosis in cancer cells remains unclear. We determined the apoptosome activity and p53 status of normal human cells and of lung, colon, stomach, brain, and breast cancer cells by measuring cytochrome c-dependent caspase activation and by DNA sequencing, respectively, and we used COMPARE analysis to identify apoptosome-specific agonists. We compared cell death, cytochrome c release, and caspase activation in NCI-H23 (lung cancer), HCT-15 (colon cancer), and SF268 (brain cancer) cells treated with Triacsin c, an inhibitor of acyl-CoA synthetase (ACS), or with vehicle. The cells were mock, transiently, or stably transfected with genes for Triacsin c-resistant ACSL5, dominant negative caspase-9, or apoptotic protease activating factor-1 knockdown. We measured ACS activity and levels of cardiolipin, a mitochondrial phospholipid, in mock and ACSL5-transduced SF268 cells. Nude mice carrying NCI-H23 xenograft tumors (n = 10) were treated with Triacsin c or vehicle, and xenograft tumor growth was assessed. Groups were compared using two-sided Student t tests. Of 21 p53-defective tumor cell lines analyzed, 17 had higher apoptosome activity than did normal cells. Triacsin c selectively induced apoptosome-mediated death in tumor cells (caspase activity of Triacsin c-treated versus untreated SF268 cells; means = 1020% and 100%, respectively; difference = 920%, 95% CI = 900% to 940%; P<.001). Expression of ACSL5 suppressed Triacsin c-induced cytochrome c release and subsequent cell death (cell survival of Triacsin c-treated mock- versus ACSL5-transduced SF268 cells; means = 40% and 83%, respectively; difference = 43%, 95% CI = 39% to 47%; P<.001). ACS was also essential to the maintenance of cardiolipin levels. Finally, Triacsin c suppressed growth of xenograft tumors (relative tumor volume on day 21 of Triacsin c-treated versus untreated mice; means = 4.6 and 9.6, respectively; difference = 5.0, 95% CI = 2.1 to 7.9; P = .006). Many p53-defective tumors retain activity of the apoptosome, which is therefore a potential target for cancer chemotherapy. Inhibition of ACS may be a novel strategy to induce the death of p53-defective tumor cells.

Small Interfering RNA–Mediated Polo-Like Kinase 1 Depletion Preferentially Reduces the Survival of p53-Defective, Oncogenic Transformed Cells and Inhibits Tumor Growth in Animals

Polo-like kinase 1 (Plk1) is required for multiple stages of mitosis and is up-regulated in many human malignancies. We depleted Plk1 expression using small interfering RNA (siRNA) and showed defects in bipolar spindle formation and cytokinesis, growth inhibition, and apoptosis induction in human cancer cell lines. To our surprise, depletion of Plk1 in normal human cells did not result in obvious cell cycle defects, and did not induce significant inhibition of cell growth for at least two cell cycles. In addition, Plk1 siRNA inhibited colony formation in soft agar and tumorigenesis in a HT1080 xenograft model in a dose-dependent manner. Analysis with isogenic pairs of cell lines, differing in p53 status, revealed that Plk1 depletion preferentially induced mitotic arrest, aneuploidy, and reduced cell survival in the p53-defective cell lines. No obvious defects were observed in most p53 wild-type cells during the first few cell cycles. In addition, long-term survival studies revealed that p53 facilitates survival upon Plk1 depletion. Therefore, short-term inhibition of Plk1 can kill tumor cells while allowing normal cells to survive. These data validate the episodic inhibition of Plk1 as a very useful approach for cancer treatment.

Repression of mRNA for the PLK cell cycle gene after DNA damage requires BRCA1.

DNA damage activates the G2 cell cycle checkpoint to allow time for DNA repair before mitotic entry. The mechanism involves inhibition of the enzymatic activity for polo-like kinase 1 (Plk1), rendering Cdc25C with a basal phosphatase activity that is insufficient for converting Cdc2 to the fully active G2/M transition kinase. We found that cell cycle arrest at the G2/M boundary after ionizing radiation (IR) of breast carcinoma cells may involve repression of the gene for Plk1, PLK, mediated by the tumor-suppressor protein BRCA1. The p53-defective MT-1 cell line had an apparent accumulation of G2/M phase cells 12 h after irradiation. This response was preceded by a transient downregulation of PLK mRNA expression with a barely detectable level 6 h after exposure to IR but recovered after 12 h. A significantly lower fraction of irradiated BRCA1(-/-) HCC1937 cells arrested in the G2/M phase after 12 h, and the transient response of PLK mRNA was also considerably impaired. After reconstitution of wild-type BRCA1 in the HCC1937 cells however, downregulation of PLK mRNA as well as Plk1 protein expression after IR was restored. Moreover, the suppression of PLK mRNA expression 6 h after irradiation was completely abolished by the specific CHEK1 kinase inhibitor UCN-01, further indicating that the effector mechanism of DNA damage on PLK signals through BRCA1 and its downstream CHEK1. Our observations provide new information about the diversity of regulatory mechanisms governed by BRCA1 in DNA damage checkpoint control.

Overexpression of mammalian Rad51 does not stimulate tumorigenesis while a dominant-negative Rad51 affects centrosome fragmentation, ploidy and stimulates tumorigenesis, in p53-defective CHO cells.

Rad51 protein plays a pivotal role in homologous recombination (HR), which is involved in double-strand break repair and in genome maintenance. Despite interactions with tumor suppressor proteins, the role of mammalian Rad51 and more generally of HR in tumor prevention is not clearly established. Indeed, both high and low frequencies of HR as well as high and low levels of RAD51 expression have been reported in tumors and in precancerous conditions. To address the question of the impact of HR on tumorigenesis, we used Chinese hamster ovary (CHO) p53-defective cell lines overexpressing the mouse MmRAD51, which stimulates HR (we name these lines: Hyper-rec lines). In parallel, we used CHO cell lines expressing a RAD51 dominant-negative form that specifically inhibits gene conversion without affecting cell viability (Hypo-rec lines). These different lines were injected into nude mice to measure their tumorigenicity. Hypo-rec lines generated a higher frequency of tumors, which also exhibited faster growth, compared to control and Hyper-rec lines. Consistent with tumorigenicity, Hypo-rec cells exhibit spontaneous centrosome duplication defects and aneuploidy. These results are the first direct evidence of involvement of RAD51 in tumor repression.

Functional inactivation of p73, a homolog of p53 tumor suppressor protein, by human papillomavirus E6 proteins

Human papillomavirus (HPV) is strongly implicated as a causative agent in the etiology of cervical cancer. Of its gene products, E6 binds to and inactivates p53 tumor suppressor protein by ubiquitin/proteasome-dependent degradation. Recently, p73, a novel family of p53, has been identified and demonstrated, like p53, to activate p21WAF1. Here we show that p73 is also inactivated by HPV-E6, but ubiquitin-mediated proteolysis is not responsive. Yeast two-hybrid and GST pull-down assays indicate a physical interaction between p73 and either HPV-16 or HPV-11 E6 proteins in vivo and in vitro, respectively. The transactivation domain (amino acid residues 1 to 49) is found to be absolutely required for the interaction. Transient co-expression of E6 significantly inhibits the p73-mdiated activation of p21WAF1 promoter in a p53-defective C33A cell line. Using Gal4-p73 fusion protein, we demonstrate that E6 inhibition of p73 transactivation function is independent of sequence-specific DNA binding, which is confirmed by a direct electrophoretic mobility shift assay. Moreover, E6 inhibits p73 function by interfering with the activity of the amino-terminal activation domain. Co-transfection of E6 mutants reveals that the same portion of E6 appears to be responsible for the inactivation of p53 and p73 function. However, the inactivation mechanism of p73 is clearly different from that of p53, because p73, unlike p53, is inactivated by both high- and low-risk E6s and is not susceptible to E6-dependent proteolysis. These overall results, consequently, suggest that in addition to the inactivation of p53, the functional interference of p73 by HPV-E6 may, at least in part, contribute to E6-mediated transformation and hyperproliferation of cervical cells. © 2001 Wiley-Liss, Inc.

Functional inactivation of p73, a homolog of p53 tumor suppressor protein, by human papillomavirus E6 proteins.

Human papillomavirus (HPV) is strongly implicated as a causative agent in the etiology of cervical cancer. Of its gene products, E6 binds to and inactivates p53 tumor suppressor protein by ubiquitin/proteasome-dependent degradation. Recently, p73, a novel family of p53, has been identified and demonstrated, like p53, to activate p21(WAF1). Here we show that p73 is also inactivated by HPV-E6, but ubiquitin-mediated proteolysis is not responsive. Yeast two-hybrid and GST pull-down assays indicate a physical interaction between p73 and either HPV-16 or HPV-11 E6 proteins in vivo and in vitro, respectively. The transactivation domain (amino acid residues 1 to 49) is found to be absolutely required for the interaction. Transient co-expression of E6 significantly inhibits the p73-mdiated activation of p21(WAF1) promoter in a p53-defective C33A cell line. Using Gal4-p73 fusion protein, we demonstrate that E6 inhibition of p73 transactivation function is independent of sequence-specific DNA binding, which is confirmed by a direct electrophoretic mobility shift assay. Moreover, E6 inhibits p73 function by interfering with the activity of the amino-terminal activation domain. Co-transfection of E6 mutants reveals that the same portion of E6 appears to be responsible for the inactivation of p53 and p73 function. However, the inactivation mechanism of p73 is clearly different from that of p53, because p73, unlike p53, is inactivated by both high- and low-risk E6s and is not susceptible to E6-dependent proteolysis. These overall results, consequently, suggest that in addition to the inactivation of p53, the functional interference of p73 by HPV-E6 may, at least in part, contribute to E6-mediated transformation and hyperproliferation of cervical cells.

Expression of protein kinase C beta1 confers resistance to TNFalpha- and paclitaxel-induced apoptosis in HT-29 colon carcinoma cells.

The expression of different protein kinase C (PKC) isoenzymes has been shown to vary with proliferation rates, differentiation or apoptosis in normal colon crypts. In addition, the activity of some PKC isoenzymes appears to be reduced in colorectal cancer. The aim of the present work was to determine whether modulation of PKC expression would affect the susceptibility of a p53-defective colon carcinoma cell line to different apoptotic treatments. HT-29 cells exhibited sensitivity to paclitaxel (Taxol) and tumor necrosis factor alpha (TNFalpha) in a dose- and time-dependent manner but were relatively resistant to etoposide. Inhibition of PKC activity augmented the susceptibility of HT-29 cells to apoptosis, and phorbol ester induction of PKC reduced such susceptibility. Transfected HT-29(PKC) cells, hyper-expressing the beta1 isoform of PKC, were less sensitive to TNFalpha and paclitaxel than the normal counterpart. The present data 1) indicate that the expression of PKC influences the susceptibility of HT-29 colon cancer cells to apoptotic drugs apparently regardless of their mechanism of action, and 2) suggest paclitaxel as a potential candidate for the treatment of colon cancer, possibly in association with inhibitors of PKC (alpha and beta) at doses not cytotoxic per se.