Patrick M. O'Connor

Polo-like Kinase Is a Cell Cycle-regulated Kinase Activated during Mitosis

Previously, we demonstrated that expression of polo-like kinase (PLK) is required for cellular DNA synthesis and that overexpression of PLK is sufficient to induce DNA synthesis. We now report that the endogenous levels of PLK, its phosphorylation status, and protein kinase activity are tightly regulated during cell cycle progression. PLK protein is low in G1, accumulates during S and G2M, and is rapidly reduced after mitosis. During mitosis, PLK is phosphorylated on serine, and its serine threonine kinase function is activated at a time close to that of p34. The phosphorylated form of PLK migrates with reduced mobility on SDS-polyacrylamide gel electrophoresis, and dephosphorylation by purified protein phosphatase 2A converts it to the more rapidly migrating form and reduces the total amount of PLK kinase activity. Purified p34-cyclin B complex can phosphorylate PLK protein in vitro but causes little increase in PLK kinase activity.

Cells lacking CIP1/WAF1 genes exhibit preferential sensitivity to cisplatin and nitrogen mustard.

We have previously shown that p53 disruption sensitizes certain cancer cell types to cisplatin (CDDP) (Fan et al., 1995). In the present study we investigated the role of the p53 downstream effector, p21CIP1/WAF1 (p21), in this sensitization. Studies were performed in human colon cancer HCT-116 cells and murine embryonic fibroblasts (MEF) with intact versus disrupted p21 genes. For comparison, HCT-116 cells lacking p53 function were also prepared through stable transfection with the human papillomavirus type-16 E6 gene. HCT-116/E6 cells were found to be more sensitive than control transfectants to CDDP and another DNA crosslinking agent, nitrogen mustard (HN2). HCT-116 cells with disrupted p21 genes also exhibited greater CDDP and HN2-sensitivity than parental HCT-116 cells. In contrast, the clonogenic survival of HCT-116 cells exposed to ionizing radiation, adriamycin, taxol or vincristine was not affected by p53 or p21 disruption. Sensitization of HCT-116/p21−/− cells to CDDP and HN2 was not limited to the HCT-116 cell background since MEF from p21 knockout mice were also more sensitive to these DNA crosslinking agents. Investigations into a possible cause of this enhanced sensitivity revealed that HCT-116 cells lacking p53 or p21 function exhibited a reduced ability to repair cisplatin-damaged CAT-reporter plasmids transfected into the cells. In addition, we found that HCT-116/p21−/− cells were much more susceptible to HN2-induced cell cycle delay than parental cells. Our results suggest that p21 disruption preferentially sensitizes at least some cell types to DNA crosslinking agents.

Sequential administration of camptothecin and etoposide circumvents the antagonistic cytotoxicity of simultaneous drug administration in slowly growing human colon carcinoma HT-29 cells

We compared the cytotoxicity of simultaneous and sequential combination chemotherapy with camptothecin and etoposide, in slowly growing human colon carcinoma, HT-29 cells. Simultaneous treatments of HT-29 cells with etoposide and camptothecin produced no marked enhancement of cytotoxicity over single agent administration. This finding demonstrates antagonism of one drugs cytotoxicity over the other. When these studies were repeated in sequential treatment protocols, we observed that antagonism could be circumvented if the period between individual drug administration was separated by 6-8 h. The cytotoxicity that was observed with this approach was never more than additive and the order of camptothecin or etoposide administration did not significantly affect the extent of combined cytotoxicity observed. The protective effect of simultaneous camptothecin and etoposide exposure was not due to reduced formation or alterations in the rate of cleavable complex reversal, and protection persisted for a considerably longer period of time than DNA strand breaks. Protection correlated with the kinetics of DNA and RNA synthesis inhibition produced by either drug. Remarkably, full cytotoxic protection could be afforded by one drug over the other, in the presence of only partial inhibition of DNA or RNA synthesis (50-60%). Our findings suggest that sequential rather than simultaneous administration of topoisomerase I and II inhibitors in future cancer chemotherapy schedules will enhance cytotoxicity over single-agent administration.

Mining the NCI Anticancer Drug Discovery Databases: Genetic Function Approximation for the QSAR Study of Anticancer Ellipticine Analogues

The U.S. National Cancer Institute (NCI) conducts a drug discovery program in which approximately 10,000 compounds are screened every year in vitro against a panel of 60 human cancer cell lines from different organs of origin. Since 1990, approximately 63,000 compounds have been tested, and their patterns of activity profiled. Recently, we analyzed the antitumor activity patterns of 112 ellipticine analogues using a hierarchical clustering algorithm. Dramatic coherence between molecular structures and activity patterns was observed qualitatively from the cluster tree. In the present study, we further investigate the quantitative structure-activity relationships (QSAR) of these compounds, in particular with respect to the influence of p53-status and the CNS cell selectivity of the activity patterns. Independent variables (i.e., chemical structural descriptors of the ellipticine analogues) were calculated from the Cerius2 molecular modeling package. Important structural descriptors, including partial atomic charges on the ellipticine ring-forming atoms, were identified by the recently developed genetic function approximation (GFA) method. For our data set, the GFA method gave better correlation and cross-validation results (R2 and CVR2 were usually approximately 0.3 higher) than did classical stepwise linear regression. A procedure for improving the performance of GFA is proposed, and the relative advantages and disadvantages of using GFA for QSAR studies are discussed.

Methods for Synchronizing Cells at Specific Stages of the Cell Cycle

Exponentially growing cells are asynchronous with respect to the cell cycle stage. Detection of cell cycle‐related events is improved by enriching the culture for cells at the stage during which the particular event occurs. Methods for synchronizing cells are provided here, including those based on morphological features of the cell (mitotic shake‐off), cellular metabolism (thymidine inhibition, isoleucine depravation), and chemical inhibitors of cell progression in G1 (lovastatin), S (aphidicolin, mimosine), and G2/M (nocodazole). Applications of these methods and the advantages and disadvantages of each are described.

Relationships between proto-oncogene expression and apoptosis induced by anticancer drugs in human prostate tumor cells

A variant of human prostate PC3 cells, isolated from PC3 cells, was shown to be significantly resistant (> 10-fold) to several clinically active anticancer drugs, including VP-16 and cisplatin. Previous studies showed that resistance to these drugs was not due to expression of the mdr1 gene, or modifications in topoisomerases but may have resulted from high expressions of certain proto-oncogenes (Yamazaki et al. (1994) Biochim. Biophys. Acta 1226, 89-96). Flow cytometry, DNA gel electrophoresis and northern blot analysis were used to further characterize drug responses in sensitive and resistant cells. Treatment of the sensitive PC3 cells with VP-16 and CDDP resulted in accumulation of cells in S and G2, and G1 and S phases, respectively, and caused significant degradation of the genomic DNA into internucleosomal sized DNA fragments, indicating apoptosis. In contrast, resistant PC3 cells showed little or no DNA fragmentation. Resistant PC3(R) cells expressed 2-3-fold more bcl2 protein than the parental PC3 cells, and overexpressed c-myc, c-jun and H-ras mRNA compared to sensitive cells. Treatment with VP-16 or CDDP significantly induced c-myc mRNA levels in sensitive PC3 cells. H-ras message was not affected by either VP-16 or CDDP treatment in PC3 cells. These studies, taken together, suggest that a differential susceptibility to apoptosis and chemosensitivity may be related to altered levels of bcl2 and/or oncogene overexpression in PC3(R) cells.

RPR-115135, a farnesyltransferase inhibitor, increases 5-FU- cytotoxicity in ten human colon cancer cell lines: Role of p53

A new non peptidic farnesyltransferase inhibitor, RPR‐115135, in combination with 5‐FU was studied in 10 human colon cancer cell lines (HCT‐116, RKO, DLD‐1, Colo‐320, LoVo, SW‐620, HT‐29, HCT‐15, Colo‐205 and KM‐12) carrying several mutations but well characterized for p53 and Ras status. We found that there was a slight tendency (not statistically significant) for the p53 inactivated cells to be less sensitive to 5‐FU after 6 days continuous treatment. Simultaneous administration of RPR‐115135 and 5‐FU, at subtoxic concentrations, resulted in a synergistic enhancement of 5‐FU cytotoxicity in the p53 wildtype cells (HCT‐116, RKO, DLD‐1, Colo‐320, LoVo). In the p53 mutated cells (SW‐620, HT‐29, HCT‐15, Colo‐205, KM‐12) the effect was very complicated. In HCT‐15 the combination resulted in antagonism, in KM‐12 in antagonism or in synergy (at different concentrations) and in SW‐620, HT‐29 and Colo‐205 cells in synergy but only when 5‐FU was administered at high concentrations. Growth inhibition could be accounted for on the basis of a specific cell cycle arrest phenotype (G2‐M arrest), as assayed by flow cytometry, only in the p53 functioning cell lines. The combination RPR‐115135 + 5‐FU increases apoptotic events only in these cell lines. In the mutated cell lines no major alterations on cell cycle arrest phenotype and no induction of apoptosis was observed. Although RPR‐115135 can potentiate the effect of 5‐FU in cells in which p53 function is disrupted, these data suggest strongly that RPR‐115135 significantly enhances the efficacy of 5‐FU only when p53 is functioning.

S-phase population analysis does not correlate with the cytotoxicity of camptothecin and 10,11-methylenedioxycamptothecin in human colon carcinoma HT-29 cells

Previous studies in rapidly proliferating rodent cells have suggested that the lethal effect of the DNA topoisomerase I inhibitor, camptothecin (CPT) is dependent upon the active participation of DNA replication (Holm et al. Cancer Res. 49:6365-6368; 1989). The purpose of the current study was to determine if this relationship applies to more slowly growing human cells. In our present study, we employed the human colon carcinoma cell line, HT-29 (45 hr doubling time). Flow cytometric determination of S-phase cells either by S-phase fit model or rectangle fit model analysis predicted that 21% of exponentially growing HT-29 cells were undergoing DNA replication. These findings were confirmed by immunofluorescence microscopy of bromodeoxyuridine labeled cells. Based on these findings, we would have expected only 20-30% of the cells to be susceptible to brief treatment (30 min) with CPT. Instead, 90-95% of HT-29 cells were killed. This apparent disparity was not due to prolonged cellular retention of drug after treatment because protein-linked DNA strand breaks reversed within 15 min of drug removal. Moreover, the DNA replication inhibitor, aphidicolin, fully protected HT-29 cells against CPT-induced killing but did not affect the production of CPT-induced protein-linked DNA strand breaks. Similar results were also obtained using the CPT-analog, 10,11-methylenedioxy-camptothecin, which was 5- to 10-fold more potent than camptothecin (OConnor et al. Cancer Commun. 2:395-400; 1990).(ABSTRACT TRUNCATED AT 250 WORDS)

10,11-Methylenedioxycamptothecin, a Topoisomerase I Inhibitor of Increased Potency: DNA Damage and Correlation to Cytotoxicity in Human Colon Carcinoma (HT-29) Cells

We had previously shown that 10,11-methylenedioxy-20-(RS)-camptothecin (MDO-CPT) is a more potent inhibitor of purified DNA topoisomerase I than 20-(S)-camptothecin (CPT). The current studies compared the cytotoxicity and DNA damage induced by MDO-CPT and CPT in the human colon carcinoma cell line, HT-29. MDO-CPT was 7- to 10-fold more potent than CPT both for cytotoxicity (ID50 = 25 vs. 180 nM) and production of DNA single-strand breaks (SSB). Kinetics of SSB formation and reversal were similar for MDO-CPT and CPT. DNA-protein crosslinks (DPC) were also produced by both drugs with a SSB/DPC ratio of 1/1. Moreover, no SSB were detected under non-deproteinizing conditions, indicating that both CPT and MDO-CPT produced protein-linked DNA single-strand breaks. A good correlation between cytotoxic potency and protein-linked DNA single-strand break production was observed for CPT and MDO-CPT, implying a causal relationship between drug-induced cytotoxicity and topoisomerase I inhibition. The sensitivity of human colon HT-29 cancer cells to camptothecins may be a selective phenomenon since these cells normally express natural resistance to current chemotherapeutic drugs, including topoisomerase II inhibitors.

Cellular Effects of a New Farnesyltransferase Inhibitor, RPR-115135, in a Human Isogenic Colon Cancer Cell Line Model System HCT-116

P. RUSSO,a,d,f C. OTTOBONI,a,d C. FALUGI,b W. REINHOLD,a J.F. RIOU,c S. PARODI,d AND P.M. O’CONNORa,e aLaboratory of Molecular Pharmacology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA bDepartment of Developing Biology, University of Genoa, Genoa, Italy cAnticancer Research Program, Centre de Recherche Rhone-Poulenc Rorer, Vitry F-94403, France dLaboratory of Experimental Oncology, National Cancer Institute and Department of Clinical and Experimental Oncology, University of Genoa, Genoa I-16132, Italy