W. Robert Bishop

Human survivin is negatively regulated by wild-type p53 and participates in p53-dependent apoptotic pathway

Survivin is an inhibitor of apoptosis protein, which is over-expressed in most tumors. Aberrant expression of survivin and loss of wild-type p53 in many tumors prompted us to investigate a possible link between these two events. Here we show that wild-type p53 represses survivin expression at both mRNA and protein levels. Transient transfection analyses revealed that the expression of wild-type p53, but not mutant p53, was associated with strong repression of the survivin promoter in various cell types. The over-expression of exogenous survivin protein rescues cells from p53-induced apoptosis in a dose-dependent manner, suggesting that loss of survivin mediates, at least, in part the p53-dependent apoptotic pathway. In spite of the presence of two putative p53-binding sites in the survivin promoter, deletion and mutation analyses suggested that neither site is required for transcriptional repression of survivin expression. This was confirmed by chromatin immunoprecipitation assays. Further analyses suggested that the modification of chromatin within the survivin promoter could be a molecular explanation for silencing of survivin gene transcription by p53.

Farnesyl Transferase Inhibitors Block the Farnesylation of CENP-E and CENP-F and Alter the Association of CENP-E with the Microtubules

Human tumor cell lines that are sensitive to the effects of farnesyl transferase inhibitors accumulate in G2 → M (except for cells with an activated Ha-ras that accumulate in G1). A search for CAAX box proteins from Swiss-Prot revealed more than 300 peptides. Of these, the centromeric proteins CENP-E and CENP-F are preferentially expressed during mitosis and are implicated as mediators of the G2 → M checkpoint. Experiments performed here show that peptides from the COOH-terminal CAAX box of CENP-E and CENP-F are substrates for farnesyl transferase but not geranylgeranyl transferase-I. Although both proteins are prenylated in the human tumor cell line DLD-1, their prenylation is completely inhibited by the farnesyl transferase inhibitor, SCH 66336. Immunohistochemical data with the lung carcinoma cell line, A549, showed that preventing the farnesylation of CENP-E and CENP-F by treatment with the farnesyl transferase inhibitor SCH 66336 does not affect their localization to the kinetochores. However, the presence of farnesyl transferase inhibitors alters the association between CENP-E and the microtubules. Our results imply that the inhibition of CENP-E farnesylation results in the alteration of the microtubule-centromere interaction during mitosis and results in the accumulation of cells prior to metaphase.

Global transcriptional program of p53 target genes during the process of apoptosis and cell cycle progression

The temporal gene expression profile during the entire process of apoptosis and cell cycle progression in response to p53 in human ovarian cancer cells was explored with cDNA microarrays representing 33 615 individual human genes. A total of 1501 genes (4.4%) were found to respond to p53 (approximately 80% of these were repressed by p53) using 2.5-fold change as a cutoff. It was anticipated that most of p53 responsive genes resulted from the secondary effect of p53 expression at late stage of apoptosis. To delineate potential p53 direct and indirect target genes during the process of apoptosis and cell cycle progression, microarray data were combined with global p53 DNA-binding site analysis. Here we showed that 361 out of 1501 p53 responsive genes contained p53 consensus DNA-binding sequence(s) in their regulatory region, approximately 80% of which were repressed by p53. This is the first time that a large number of p53-repressed genes have been identified to contain p53 consensus DNA-binding sequence(s) in their regulatory region. Hierarchical cluster analysis of these genes revealed distinct temporal expression patterns of transcriptional activation and repression by p53. More genes were activated at early time points, while more repressed genes were found after the onset of apoptosis. A small-scale quantitative chromatin immunoprecipitation analysis indicated that in vivo p53–DNA interaction was detected in eight out of 10 genes, most of which were repressed by p53 at the early onset of apoptosis, suggesting that a portion of p53 target genes in the human genome could be negatively regulated by p53 via sequence-specific DNA binding. The approaches and genes described here should aid the understanding of global gene regulatory network of p53.

Differential effects of cell cycle regulatory protein p21WAF1/Cip1 on apoptosis and sensitivity to cancer chemotherapy

p21(WAF1/Cip1) was initially identified as a cell cycle regulatory protein that can cause cell cycle arrest. It is induced by both p53-dependent and p53-independent mechanisms. This mini-review briefly discusses its currently known functions in apoptosis and drug sensitivity. As an inhibitor of cell proliferation, p21(WAF1/Cip1) plays an important role in drug-induced tumor suppression. Nevertheless, a number of recent studies have shown that p21(WAF1/Cip1) can assume both pro- or anti-apoptotic functions in response to anti-tumor agents depending on cell type and cellular context. This dual role of p21(WAF1/Cip1) in cancer cells complicates using p21(WAF1/Cip1) status to predict response to anti-tumor agents. However, it is possible to develop p21(WAF1/Cip1)-targeted reagents or p21(WAF1/Cip1) gene transfer techniques to have a beneficial effect within a well-defined therapeutic context. Better understanding of the roles of p21(WAF1/Cip1) in tumors should enable a more rational approach to anti-tumor drug design and therapy.

The farnesyl protein transferase inhibitor SCH66336 synergizes with taxanes in vitro and enhances their antitumor activity in vivo

Purpose: SCH66336 is an orally active, farnesyl protein transferase inhibitor. SCH66336 inhibits ras farnesylation in tumor cells and suppresses tumor growth in human xenograft and transgenic mouse cancer models in vivo. The taxanes, paclitaxel (Taxol) and docetaxel (Taxotere) block cell mitosis by enhancing polymerization of tubulin monomers into stabilized microtubule bundles, resulting in apoptosis. We hypothesized that anticancer combination therapy with SCH66336 and taxanes would be more efficacious than single drug therapy. Methods: We tested the efficacy of SCH66336 and taxanes when used in combination against tumor cell proliferation in vitro, against NCI-H460 human lung tumor xenografts in nude mice, and against mammary tumors in wap-ras transgenic mice. Results: SCH66336 synergized with paclitaxel in 10 out of 11 tumor cells lines originating from breast, colon, lung, ovary, prostate, and pancreas. SCH66336 also synergized with docetaxel in four out of five cell lines tested. In the NCI-H460 lung cancer xenograft model, oral SCH66336 (20 mg/kg twice daily for 14 days) and intraperitoneal paclitaxel (5 mg/kg once daily for 4 days) caused a tumor growth inhibition of 56% by day 7 and 65% by day 14 compared to paclitaxel alone. Male transgenic mice of the wap-ras/F substrain [FVB/N-TgN(WapHRAS)69LlnYSJL] spontaneously develop mammary tumors at 6–9 weeks of age which have been previously shown to be resistant to paclitaxel. Paclitaxel resistance was confirmed in the present study, while SCH66336 inhibited growth of these tumors. Most importantly, SCH66336 was able to sensitize wap-ras/F mammary tumors to paclitaxel chemotherapy. Conclusion: Clinical investigation of combination therapy using SCH66336 and taxanes in cancer patients is warranted. Further, SCH66336 may be useful for sensitizing paclitaxel-resistant tumors to taxane treatment.

Discovery of novel nonpeptide tricyclic inhibitors of ras farnesyl protein transferase

A comprehensive structure-activity relationship (SAR) study of novel tricyclic amides has been undertaken. The discovery of compounds that are potent FPT inhibitors in the nanomolar range has been achieved. These compounds are nonpeptidic and do not contain sulfhydryl groups. They selectively inhibit farnesyl protein transferase (FPT) and not geranylgeranyl protein transferase-1 (GGPT-1). They also inhibit H-Ras processing in Cos monkey kidney cells.

Integrative Genomics Revealed RAI3 Is a Cell Growth-promoting Gene and a Novel P53 Transcriptional Target

In this study, differential gene expression between normal human mammary epithelial cells and their malignant counterparts (eight well established breast cancer cell lines) was studied using Incyte GeneAlbum 1–6, which contains 65,873 cDNA clones representing 33,515 individual genes. 3,152 cDNAs showed a ≥3.0-fold expression level change in at least one of the human breast cancer cell lines as compared with normal human mammary epithelial cells. Integration of breast tumor gene expression data with the genes in the tumor suppressor p53 signaling pathway yielded 128 genes whose expression is altered in breast tumor cell lines and in response to p53 expression. A hierarchical cluster analysis of the 128 genes revealed that a significant portion of genes demonstrate an opposing expression pattern, i.e. p53-activated genes are down-regulated in the breast tumor lines, whereas p53-repressed genes are up-regulated. Most of these genes are involved in cell cycle regulation and/or apoptosis, consistent with the tumor suppressor function of p53. Follow-up studies on one gene, RAI3, suggested that p53 interacts with the promoter of RAI3 and repressed its expression at the onset of apoptosis. The expression of RAI3 is elevated in most tumor cell lines expressing mutant p53, whereas RAI3 mRNA is relatively repressed in the tumor cell lines expressing wild-type p53. Furthermore, ectopic expression of RAI3 in 293 cells promotes anchorage-independent growth and small interfering RNA-mediated depletion of RAI3 in AsPc-1 pancreatic tumor cells induces cell morphological change. Taken together, these data suggest a role for RAI3 in tumor growth and demonstrate the predictive power of integrative genomics.

A cembranolide diterpene farnesyl protein transferase inhibitor from the marine soft coral Lobophytum cristagalli

Abstract A previously described cembranolide diterpene from Lobophytum cristagalli was identified as a potent (IC50 0.15 μM) inhibitor of farnesyl protein transferase (FPT). The compound showed selectivity for FPT as compared to the closely related enzyme geranylgeranyl protein transferase-1 (IC50 5.3 μM). Kinetic evaluation suggests that this compound competes with the protein/peptide farnesyl acceptor substrate, and not with farnesyl pyrophosphate for inhibition of FPT.

NOVEL TRICYCLIC AMINOACETYL AND SULFONAMIDE INHIBITORS OF RAS FARNESYL PROTEIN TRANSFERASE

Abstract Novel tricyclic FPT inhibitors with submicromolar FPT activity are described. Greatly enhanced FPT activity is realized with phthaloyl derivatized amino compound 2k, which showed FPT inhibitory activity of IC50 = 0.66 μM. Sulfonamides 5g and 50 were also found to be potent FPT inhibitor. SAR resulting from a variety of tricyclic amino acids and sulfonamide derivatives is discussed.

Usnic acid amide, a phytotoxin and antifungal agent from Cercosporidium henningsii

Abstract Usnic acid amide, a phytotoxin related to (−)-usnic acid and (−)-cercosporamide, has been isolated from the cassava fungal pathogen Cercosporidium henningsii . Usnic acid amide also shows moderate antifungal activity and inhibiton of protein kinase C.