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Studies into the molecular mechanisms of cancer have revealed that, with a few exceptions, the disease lacks a specific drug target. Therefore, new anticancer drugs not only take many years and much money to develop but also might not outperform existing drugs. However, what are assumed to be unfavorable hallmarks of cancer, for example drug resistance, can be exploited for selective anticancer therapy and for protection of normal cells. Based on this paradigm, the drug discovery can be complemented by novel use of existing agents and even "failed" drugs. The pharmacological industry could develop low cost, effective therapeutic modalities, by "re-using" already marketed and late-stage products in cancer-selective therapeutic kits.

A phase III failure of Iressa, an inhibitor of the EGF receptor (EGFR), is viewed as a surprise. With a few exceptions, however, inhibitors of EGFR cannot be effective as a monotherapy in cancers with additional oncogenic changes (downstream of EGFR), which cause mitogen-independent proliferation. In other cases, combining these inhibitors with chemotherapy may lead to antagonism in cancer cells and/or aggravated side-effects. This review discusses why neither levels of EGFR nor doses of Iressa correlate with clinical response. We suggest a novel use of signal transduction inhibitors, including Iressa, to increase therapeutic index by modulating cycle-dependent and apoptosis-inducing chemotherapies. This approach may be most beneficial to patients who do not respond to monotherapy with kinase inhibitors. Development of molecular diagnostics will further diversify these strategies.

We have previously described a novel mechanism of p53 dysfunction, characterized by repression of mRNA and protein expression effectively leading to functional inactivation of wt p53 in SW-1736 human anaplastic thyroid cancer cells (pseudo-null p53). Here we demonstrated that treatment of SW-1736 cells with sub-cytotoxic concentrations of FR901228, a histone deacetylase (HDAC) inhibitor, results in marked induction of p53 mRNA and protein. The p53 induced by FR901228 was functional as evidenced by mdm-2 and p21 transactivation, and its further accumulation following DNA damage by doxorubicin. Furthermore, pretreatment with FR901228 sensitized SW-1736 cells to doxorubicin. This study validates the concept of pseudo-null p53, as a mechanism of p53 inactivation, and demonstrates that pseudo-null p53 can be rescued pharmacologically.

Avoidance of apoptosis and mitogen-independent growth are hallmarks of cancer. Mitogen-activated kinases (for example, ErbB1, Raf-1, MEK, PI-3-K, mTOR) can suppress chemotherapy-induced apoptosis in cancer cells. While kinase inhibitors restore susceptibility of cancer cells to apoptosis, they do not necessarily cause growth arrest in cancer cells harboring additional mutations in downstream signaling pathways such as inactivation of Rb and overexpression of c-myc. This article provides a conceptual basis for a novel use of inhibitors of mitogenic kinases. While arresting growth of normal cells, kinase inhibitors may not arrest cancer cells but instead can sensitize them to apoptosis. Following pretreatment with low doses of kinase inhibitors, the chemotherapy that predominantly induces apoptosis in cycling cells (cyclotherapy) will kill cancer cells while sparing normal cells.

By causing cytoplasmic mislocation of p27 and p21, the Akt oncogenic kinase functionally inactivates these nuclear tumor suppressor proteins. Is cytoplasmic localization of p27 and p21 simply equivalent to loss of their function or are new functions acquired in the cytoplasm? Indeed, several lines of evidence suggest that cytoplasmic p27 and p21 may be oncoproteins with antiapoptotic activities.

By inducing p53-dependent G2 arrest, the pretreatment with low concentrations of DNA damaging drugs (e.g., doxorubicin, DOX) can prevent cell death caused by microtubule-active drugs (e.g., paclitaxel, PTX), thus potentially permitting selective killing of p53-deficient cancer cells. However, DOX still protects a subset of tumor cell lines lacking wt p53 (HL60 and Jurkat leukemia cells), thus limiting the utility of protection of cells with wt p53 (e.g., normal cells). The present work overcomes this obstacle by adding an abrogator of p53-independent checkpoint (e.g., UCN-01) to the DOX-PTX sequence. By inhibiting a p53-independent pathway, UCN-01 overrode DOX-induced G2 arrest and instead induced G1 arrest in HL60 and Jurkat, thus propelling these p53-deficient cells from G2 to G1. Once they entered mitosis, cells were killed by PTX. Induction of G2 arrest with sequential abrogation of a p53-independent checkpoint allows pharmacological manipulation of Raf-1/Bcl-2 hyperphosphorylation, PARP and Rb cleavage and cell death caused by PTX in p53-deficient cells. Unlike previous approaches, this strategy is intended to increase selectivity, not the cytotoxicity of PTX. This rational sequence of agents that induces p53-dependent and abrogates p53-independent arrest represents a cancer-selective strategy for treatment of p53-deficient tumors.

By preventing deacetylation of histones, histone deacetylase inhibitors (HDIs) transcriptionally induce p21. Here we show that the HDIs sodium butyrate (Bu), trichostatin A (TSA) and depsipeptide (FR901228) all induced p21, but only TSA and FR901228 caused mitotic arrest (in addition to arrest in G1 and G2). The ability to cause mitotic arrest correlated with the higher cytotoxicity of these compounds. Although causing mitotic arrest, TSA and FR901228 (unlike paclitaxel) did not affect tubulin polymerization. Unlike FR9012208, TSA caused acetylation of tubulin at lysine 40; both soluble tubulin and microtubules were acetylated. Whereas the induction of p21 reached a maximum by 8 h, tubulin was maximally acetylated after only 1 h of TSA treatment. Tubulin acetylation was detectable after treatment with 12-25 ng/ml TSA although acetylation plateaued at 50 ng/ml TSA, coinciding with G2-M arrest, appearance of cells with a sub-2N DNA content, poly(ADP-ribose) polymerase cleavage, and rapid cell death. We conclude that HDIs have differential effects on non-histone deacetylases and that rapid acetylation of tubulin caused by TSA is a marker of nontranscriptional effects of TSA.

The tumor suppressor protein p53 localizes to microtubules (MT) and, in response to DNA damage, is transported to the nucleus via the MT minus-end-directed motor protein dynein. Dynein is also responsible for MT-mediated nuclear targeting of adenovirus type 2 (Ad2). Here we show that treatment with low concentrations of MT-targeting compounds (MTCs) that do not disrupt the MT network but are known to suppress MT dynamics enhanced p53 nuclear accumulation, and the activation of the p53-downstream target genes. p53 nuclear accumulation required binding of MTCs to MTs and enhanced the induction of p53-up-regulated modulator of apoptosis (PUMA) mRNA and apoptosis on challenging cells with the DNA-damaging drug adriamycin. Low concentrations of MTCs enhanced the rate of movement of fluorescent Ad2 to the nucleus and increased the nuclear targeting efficiency of Ad2. We propose that suppression of MT dynamics by low concentrations of MTCs enhances MT-dependent trafficking toward the minus ends of MTs and facilitates nuclear targeting.

Resting cells are relatively resistant to microtubule-active drugs including paclitaxel (PTX). By causing p53-mediated arrest, pretreatment with low concentrations of doxorubicin (DOX) protected HCT116 cells from the cytotoxicity caused by PTX. Unlike DOX, flavopiridol (FL) did not protect HCT116 cells. Low concentrations of FL (50 nM) induced p21 but not p53. High concentrations of FL (500 nM) decreased levels of p21 and Mdm-2 but dramatically induced p53. Thus, FL reciprocally affects p21 and p53. In LNCaP, a prostate cancer cell line which is highly sensitive to p21-induced growth arrest (p21-sensitive), low concentrations of FL (50 nM) induced p21 (without induction of p53) and caused G1 and G2 arrest. This precluded mitotic arrest, Bcl-2 and Raf-1 phosphorylation, and diminished cell death caused by PTX. In contrast, FL did not protect PC3M, arrest-resitant and highly aggressive prostate cancer cells. Like LNCaP, HL60 and SKBr3 cells are known to be p21-sensitive. As predicted, low concentrations of FL antagonized PTX-mediated cytotoxicity in HL60 and SKBr3 cell lines. In summary, only low concentrations of FL can induce p21, and, in turn, only p21-sensitive cells are protected from PTX.

The p53 tumor suppressor can induce growth arrest, apoptosis and cell senescence. Not surprisingly, p53 is an appealing target for therapeutic intervention. Although current anticancer agents do not directly interact with p53, these agents (including DNA damaging drugs, antimetabolites, microtubule-active drugs and inhibitors of the proteasome) cause accumulation of wt p53. Depending on the p53 status of cancer cells, diverse therapeutic strategies are under development. These include pharmacological rescue of mutant p53 function and reactivation of wt p53 in E6-expressing cells. For protection of normal cells, strategies range from abrogation of wt p53 induction, thereby decreasing the toxicity of DNA damaging agents, to activation of wt p53-dependent checkpoints, thereby protecting cells against cell cycle-dependent therapeutics., (Copyright 2001 Wiley-Liss, Inc.)

Purpose: The primary objectives of this trial were to define the maximum tolerated dose (MTD) and to characterize the toxicities and pharmacokinetics of depsipeptide (FR901228) given on a day-1 and day-5 schedule every 21 days. A secondary objective of the trial was to seek evidence of antineoplastic activity., Patients and Methods: Patients with advanced or refractory neoplasms received depsipeptide by a 4-h i.v. infusion on days 1 and 5 of a 21-day cycle. On the basis of preclinical data suggesting that depsipeptide may have significant cardiac toxicity, patients were treated while receiving continuous cardiac monitoring and were followed with serial cardiac enzyme determinations, electrocardiograms (ECGs), and nuclear ventriculograms (MUGA scans). The starting dose of the trial was 1 mg/m(2), and dose escalations proceeded through a total of eight dose levels to a maximum of 24.9 mg/m(2). Toxicities were graded using the National Cancer Institute common toxicity criteria, and pharmacokinetics were determined using a liquid chromatography/tandem mass spectrometry method., Results: Patients (37) received a total of 88 cycles of treatment on study (range: one to eight cycles). Dose-limiting toxicity (DLT) was observed, and the MTD exceeded at a dose of 24.9 mg/m(2). The DLTs included grade-3 fatigue (3 patients), grade-3 nausea and vomiting (1 patient), grade-4 thrombocytopenia (2 patients), and grade-4 cardiac arrhythmia (1 patient, atrial fibrillation). The MTD was defined at the seventh dose level (17.8 mg/m(2)). Reversible ST/T changes and mild reversible dysrhythmias were observed on the post-treatment ECG. There were no clinically significant changes in left ventricular ejection fraction. One patient achieved a partial response. The plasma disposition of depsipeptide was well described by a first-order, two-compartment model. The mean volume of distribution, clearance, t(1/2alpha) and t(1/2beta) at a dose of 17.8 mg/m(2) was: 8.6 liters/m(2), 11.6 liters/h/m(2), 0.42 h, and 8.1 h, respectively. The mean maximum plasma concentration at the MTD was 472.6 ng/ml (range: 249-577.8 ng/ml). Biological assays showed that the serum levels achieved could cause the characteristic cell cycle effects of this agent when serum was added to PC3 cells in culture, as well as increased histone acetylation in patient-derived peripheral blood mononuclear cells., Conclusion: The MTD of depsipeptide given on a day-1 and -5 schedule every 21 days is 17.8 mg/m(2). The DLTs are fatigue, nausea, vomiting, and transient thrombocytopenia and neutropenia. Whereas cardiac toxicity was anticipated based on preclinical data, there was no evidence of myocardial damage. However, reversible ECG changes with ST/T wave flattening were regularly observed. Biologically active serum concentrations were achieved, and 1 patient obtained a partial response. The recommended Phase II dose is 17.8 mg/m(2) administered on day 1 and 5 of a 21-day cycle.

Many carcinogens are mutagens, indicating that mutagenesis is the driving force of carcinogenesis. But what if we turn this prevailing view on its head? I will argue here that carcinogens are cytostatic and/or cytotoxic, and it is not mutation per se, but clonal selection for resistance to these antiproliferative conditions, that leads to cancer. But why aren't all cytotoxins carcinogenic?

As formulated in 1974, the concept of the restriction point of the cell cycle was based on cell biological experiments, yet allowing accurate molecular predictions and spurring a search for the restriction factor. Although cyclin D meets the criteria of the R-factor, the picture as outlined here is more interesting and far more complex. We discuss the relationship between the restriction knot and DNA damage-checkpoints. Finally, we discuss how loss of the restriction point in cancer leads to loss of checkpoint control and to insensitivity to antimitogens including some mechanism-based anticancer therapeutics.

Microtubule-active drugs, including paclitaxel (Taxol, PTX), cause mitotic arrest, and this can result in apoptosis. A recently study has reported that PTX mediates apoptosis by upregulating FasL in Jurkat and MDA-231 cells. In contrast to the previous report, we found that anti-FasL antibodies failed to inhibit PTX-induced apoptosis in Jurkat cells. In MDA-231 cells, neither FasL nor PTX induced apoptosis. In these cells, PTX caused slow cell death without activation of caspase-3 or -8 or PARP cleavage. Doxorubicin at cytostatic concentrations did not affect FasL-induced apoptosis but inhibited PTX-induced apoptosis in Jurkat cells. Following PTX-induced mitotic arrest Jurkat cells undergo apoptosis, whereas MDA-MB-231 cells exit mitosis and form multinucleated cells which then die in a slower non-apoptotic manner.

Hypoxia causes the accumulation of the transcription factor hypoxia-inducible factor 1 (HIF-1), culminating in the expression of hypoxia-inducible genes such as those for vascular endothelial growth factor (VEGF) and NDRG-1/Cap43. Previously, we have demonstrated that intracellular calcium (Ca(2+)) is required for the expression of hypoxia-inducible genes. Here we found that, unlike with hypoxia or hypoxia-mimicking conditions, the elevation of intracellular Ca(2+) neither induced the HIF-1alpha protein nor stimulated HIF-1-dependent transcription. Furthermore, the elevation of intracellular Ca(2+) induced NDRG-1/Cap43 mRNA in HIF-1alpha-deficient cells. It also increased levels of c-Jun protein, causing its phosphorylation. The protein kinase inhibitor K252a abolished c-Jun induction and activator protein 1 (AP-1)-dependent reporter expression caused by Ca(2+) ionophore or hypoxia. K252a also significantly decreased hypoxia-induced VEGF and NDRG-1/Cap43 gene expression in both human and mouse cells. Using a set of deletion VEGF-Luc promoter constructs, we found that both HIF-1 and two AP-1 sites contribute to hypoxia-mediated induction of transcription. In contrast, only AP-1 sites contributed to Ca(2+)-mediated VEGF-Luc induction. A dominant-negative AP-1 prevented Ca(2+)-dependent transcription and partially impaired hypoxia-mediated transcription. In addition, dominant-negative AP-1 diminished the expression of the NDRG-1/Cap43 gene following hypoxia. We conclude that during hypoxia, an increase in intracellular Ca(2+) activates a HIF-1-independent signaling pathway that involves AP-1-dependent transcription. Cooperation between the HIF-1 and AP-1 pathways allows fine regulation of gene expression during hypoxia.

Clinical responses to the HER1 (EGF receptor) inhibitors and HER2/neu/ErbB2 inhibitors correlate with high levels of receptor expression. However, a significant subset of patients with high receptor levels appear to be refractory to treatment. We have observed similar results in the 60 cell lines of the NCI Anti-Cancer Drug Screen using a panel of 11 selective HER1 inhibitors. As expected, low HER1-expressing cell lines were insensitive to HER1 inhibitors. In cell lines with high HER1 expression, low concentrations of HER1 inhibitors potently inhibit both HER1 phosphorylation and the mitogen-activated protein kinase (MAPK) pathway. However, this inhibition did not always correlate with cellular arrest. High HER1-expressing cell lines can be subdivided into two groups based on their sensitivity to HER1 inhibitors. In the sensitive group, receptor and growth inhibition was concordant and occurred at sub-micromolar concentrations of HER1 inhibitors. In the insensitive group, receptor inhibition occurred at a low concentration (< 1 microM) but concentrations that were ten times or higher were required for growth inhibition. Also, neither induction of p21 and cyclin D1 nor p53 status could explain the difference between sensitive and insensitive cells. Although EGF activated the MAPK pathway in all cell lines, only drug-sensitive cell lines responded to EGF (accelerated entry from G1 to S) and to HER1 inhibitors (G1 arrest) by changes in cell cycling. Furthermore, an EGF-dependent immortalized mammary epithelial cell line was extremely sensitive to a panel of HER1 inhibitors. We infer that independence from mitogen-mediated signaling confers insensitivity to HER1 inhibitors in a large subset of cancer cell lines.

Here we investigate activation of the p53 pathway by inhibition of transcription. Comparison of cells with either mutant p53 or wt p53 indicated that inhibition of p53- dependent transcription is necessary and sufficient for wt p53 accumulation. In addition to Mdm-2, p21 is required for effective p53 degradation. Transient inhibition of transcription resulted in initial downregulation of p21 and Mdm-2 leading to accumulation of wt p53. This was followed by induction of p21 and Mdm-2, normalization of p53 levels, and p21-dependent growth arrest. Although simultaneous induction of p53 and p21 could be detected by immunoblot, levels of p53 and p21 were discordant in individual cells. By inducing p21 and Mdm-2, p53 discriminates between transient and sustained inhibition of transcription. Transient inhibition results in p21-dependent growth, while sustained inhibition of transcription leads to p53-facilitated cell death. One can envision p53 as a physiological sensor of transcriptional integrity. Transient inhibition of p53- stimulated transcription by numerous stimuli including nucleotide depletion, hypoxia, UV light may be an prevalent mechanism of activation of wt p53 and its downstream pathways.

Carcinogens induce carcinogen-specific genetic instability (defects in DNA repair). According to the 'direct-selection' model, defects in DNA repair per se provide an immediate growth advantage. According to the 'associated-selection' model, carcinogens merely select for cells with adaptive mutations. Like any mutations, adaptive mutations occur predominantly in genetically unstable cells. The 'associated-selection' model predicts that carcinogen-driven selection minimizes cytotoxic but maximizes mutagenic effects of carcinogens. A purely mutagenic (neither cytotoxic, nor cytostatic) environment will favor effective DNA repair, whereas any growth-limiting conditions (telomerase deficiency, anticancer drugs) will select for genetically unstable cells. Genetic instability is a postmark of selective pressure rather than a hallmark of cancer per se. Once selected, genetic instability facilitates the development of resistance to any other growth-limiting conditions. As an example, a putative link between prior exposure to carcinogens and the ability to develop a telomerase-independent growth is discussed.

Although the Bcl-2 family members and p53 are involved in the regulation of apoptosis, the status of apoptotic machinery (eg caspases) plays a major role in determining the mode and timing of cell death. If the apoptotic machinery is lost, inhibited, or intrinsically inactivated, the "death stars", Bcl-2 and p53, may become irrelevant to cell death. In this light, high levels of Bcl-2 may indicate that downstream apoptotic pathways are still functional. This explains why Bcl-2 overexpression can be a marker of chemosensitivity and favorable prognosis in certain cancers and why retention of wild-type p53 may manifest inactivation of caspases in aggressive cancers., (Copyright 2001 John Wiley & Sons, Inc.)

Could a rational, hypothesis-driven and well-tolerated therapy drive tumor progression? This scenario can be foreseen for antiangiogenic therapy, despite it is one of the most elegant anticancer strategies. Antiangiogenic agents inhibit growth of endothelial cells resulting in tumor hypoxia and starvation which in turn inhibit tumor growth. On the other hand, it is known that hypoxia selects for a highly aggressive and metastatic cancer and is associated with unfavorable prognosis. This review attempts to reconcile these opposite notions and to revisit the thesis that antiangiogenic therapy is "resistant to resistance". The latter logical paradigm is based on the notion that endothelial cells cannot become drug resistant. Although endothelial cells may not acquire drug-resistance, cancer cells can acquire hypoxia-resistance which is also associated with the resistance to growth arrest and apoptosis as well as high metastatic potentials. Hypoxia-inducible factor (HIF-1) renders cells capable of surviving hypoxia and stimulating endothelial growth. Disruption of the HIF-1 pathway inhibits tumor growth, indicating HIF-1 as a potential anticancer target. Furthermore, inhibition of HIF-1 is a mechanism-based antiangiogenic strategy because it is the HIF-mediated response that drives tumor angiogenesis. Pharmacological approaches to HIF-1 inhibition are discussed.

Paclitaxel (PTX), a microtubule-active agent, blocks cell proliferation by inhibiting mitotic progression leading to mitotic and postmitotic arrest and cell death. Here we demonstrate for the first time that very low concentrations of PTX (3-6 nM) can completely inhibit cell proliferation without arresting cells at mitosis. At these low concentrations that are insufficient to inhibit mitotic progression, PTX induced both p53 and p21 causing G1 and G2 arrest in A549. In contrast, low PTX concentrations failed to induce G1 and G2 arrest in A549/E6 cells, that do not express p53. Furthermore, we observed that the levels of p53 and p21 induced by adriamycin and by low concentrations of PTX in A549 cells were comparable. This observation led us to conclude that low concentrations of PTX can induce p53 and p21 sufficiently to cause G1 and G2. Many other cell lines, including HCT116 cells, do not readily upregulate p53 in response to PTX, and therefore undergo exclusively mitotic and postmitotic arrest after PTX treatment. At low concentrations that do not cause mitotic arrest, PTX did not significantly inhibit proliferation of these cells. In HCT116 cells, loss of p53 (HCT/p53(-/-)) or p21 (HCT/p21(-/-)) affects both Bax and Bcl-2 expression. In cells lacking p53, levels of Bax and p21 were decreased. In cells lacking p21, levels of wt p53 were highly increased to compensate for the loss of p21. This in turn results in upregulation of Bax and downregulation of Bcl-2 resulting in an increase of the apoptotic Bax/Bcl2 ratio consistent with increased sensitivity of these cells to apoptotic stimuli. High levels of p53 and Bax/Bcl-2 ratio can also explain why loss of p21 is rarely found in human cancer.

Chemotherapy of cancer is limited by its toxicity to normal cells. On the basis of discoveries in signal transduction and cell cycle regulation, novel mechanism-based therapeutics are being developed. Although these cell cycle modulators were designed to target cancer cells, some of them can also be applied for a different purpose, i.e., to protect normal cells against the lethality of chemotherapy. Loss of sensitivity of cancer cells to cell cycle inhibitors can be exploited for selective protection of normal cells that retain this response. Indeed, inhibition of redundant or overactivated pathways (e.g., growth factor-activated pathways) or stimulation of absent pathways in cancer cells (e.g., p53, Rb, and p16) may not arrest cycling of cancer cells. But growth arrest of normal cells will then permit selective killing of cancer cells by cycle-dependent chemotherapy.

Purpose: Overexpression of P-glycoprotein (Pgp) is one mechanism of drug resistance in cancer chemotherapy. A Phase I trial was conducted using PSC 833, a Pgp antagonist, in combination with paclitaxel in patients with refractory cancer. The objective of this study was to assess the effect of PSC 833 on the metabolism of paclitaxel and characterize the differences in 6alpha-hydroxypaclitaxel pharmacokinetics. In addition, we examined the possibility of enhanced cytotoxicity of paclitaxel by the coexistence of 6alpha-hydroxypaclitaxel., Experimental Design: Patients received paclitaxel 35 mg/m(2)/day by continuous intravenous infusion (CIVI) x 4 days without PSC 833 in cycle 1 and escalating doses of paclitaxel (13.1, 17.5, or 21.3 mg/m(2)/day CIVI x 4 days) with 5 mg/kg PSC 833 by mouth every 6 h x 7 days in cycle 2. Plasma samples were analyzed for both paclitaxel and its major metabolite with high-performance liquid chromatography methods. Using human liver microsomes, we studied the effect of PSC 833 on the metabolism of paclitaxel. In addition, the in vitro cytotoxicity of 6alpha-hydroxypaclitaxel alone and in combination with paclitaxel was evaluated., Results: Twenty-one of 22 patients had a metabolite peak (6alpha-hydroxypaclitaxel) observed in the chromatogram of plasma samples from cycle 2 when they received paclitaxel in combination with PSC 833. This metabolite was not detectable in plasma obtained during the first cycle when they received paclitaxel without PSC 833. During cycle 2, the mean concentrations of 6alpha-hydroxypaclitaxel and paclitaxel were 0.10 +/- 0.074 and 0.079 +/- 0.041 microg/ml, respectively. A moderate association was observed between total bilirubin and 6alpha-hydroxypaclitaxel concentrations (P = 0.015, r = 0.52; n = 21). Human liver microsome experiments showed that a PSC 833 concentration as high as 10 microM did not affect the production of 6alpha-hydroxypaclitaxel. Paclitaxel cytotoxicity in HL60 and K562 human leukemia cells was increased in the presence of noncytotoxic concentrations of 6alpha-hydroxypaclitaxel., Conclusions: PSC 833 increases the plasma concentration of 6alpha-hydroxypaclitaxel during paclitaxel therapy. Inhibition of cytochrome P-450 3A4 by PSC 833 may explain this in part, although other mechanisms cannot be excluded.

Overexpression of ectopic mutant p53 represses wild-type p53-stimulated transcription, known as a dominant negative effect. On the other hand, overexpression of wild-type p53 can repress transcription stimulated by several transcription factors, including hypoxia-inducible factor-1 (HIF-1). Using a panel of well-characterized Arg175 p53 mutants we found that only mutants (Tyr175, Trp175, Asp175 and Phe175) which have completely lost their ability to transactivate repress wild-type p53-stimulated Bax, p21 and PG13 promoter constructs. In contrast, Asn175, Gln175, Leu175 and Pro175 mutants which partially retained transactivating functions did not exert dominant negative effects against PG13 and p21 promoter constructs. However, these latter mutants failed to activate Bax and, instead, exerted a dominant negative effect on a Bax-Luc promoter construct. We conclude that a dominant negative effect is promoter selective as a consequence of selective loss of transactivating function. Albeit less potent than wild-type p53, all Arg175 p53 mutants retained partial ability to repress HIF-1-stimulated transcription. We propose that transrepression and the dominant negative effect have similar mechanisms and may involve competition with transcription factors (wild-type p53, HIF-1, etc.) for cofactors such as p300. Thus, a p53(22/23) mutant, which is deficient in p300 binding, did not exert dominant negative effects. Like transrepression, the dominant negative effect required overexpression of mutant p53 and, therefore, is not dominant. In the presence of a wild-type p53 allele, levels of endogenous mutant p53 protein were low in heterozygous cells. Endogenous mutant p53 became overexpressed only after loss of the second p53 allele. Therefore, endogenous mutant p53s are unable to display a dominant negative effect. This explains why loss of the second p53 allele is required to eliminate p53 functions in cancer cells.

7-Hydroxystaurosporine (UCN-01), a non-selective inhibitor of protein kinase C (PKC), and phorbol ester (PMA), a PKC activator, are undergoing clinical evaluations. We investigated the effects of UCN-01 and PMA on a panel of prostate cancer cell lines. While PMA induced p21WAF1/CIP1 and arrest growth of LNCaP cancer cells (IC50 = 0.5-1 nM), aggressive cancer cell lines (DU145, PC3, and PC3M) were resistant to PMA (IC50 >5000 nM). Low concentrations (25-50 nM) of UCN-01 abrogated PMA-induced p21 and growth arrest in LNCaP cells. These low doses of UCN-01 however did not inhibit proliferation of any prostate cancer cell line. PMA-sensitive LNCaP cells were resistant to clinically relevant concentrations of UCN-01 (IC50 = 1.2 microM), but UCN-01 inhibited growth of DU145 and PC3/3M with an IC50 of 200-400 nM. For comparison, PMA-sensitive HL60 leukemia cells were sensitive to UCN-01 due to rapid apoptosis caused by UCN-01. In PMA-resistant prostate cancer cells, UCN-01 downregulated cyclin D1, induced p21, caused morphological differentiation, and G1-phase arrest leading to slow cell death without caspase activation. Importantly, normal prostate epithelial cells (PrEC) were very sensitive to both PMA (IC50 = 0.2 nM) and UCN-01. In PrEC, UCN-01 downregulated cyclin D1 and arrest growth with an IC50 less than 100 nM. We conclude that loss of sensitivity to either UCN-01 or PMA accompanies progression of prostate cancer.

The notion that wt p53 downregulates MDR-1 links p53 mutations to multidrug resistant phenotype. Alternatively, it has been envisioned that wt p53 protects cells against DNA damaging drugs by inducing MDR-1. Opposing conclusions on the relationship between MDR-1 and p53 have been predominantly based on the effects of p53 on MDR-1 promoter-constructs. We found that introduction of wt p53 slightly induced MDR-1 mRNA in three cell lines having endogenous mt p53. Wt p53-mediated induction of endogenous MDR-1 may represent a rudiment of cellular protection against toxic compounds earlier in evolution. Marked induction of p21WAF1/CIP1 (p21) mRNA was observed in all cell lines; and lower levels of wt p53 were required to induce p21 than MDR-1. Pgp was undetectable and wt p53 did not increase resistance to an MDR-1 substrate, suggesting the changes in MDR-1 mRNA may be functionally insignificant. Unlike endogenous MDR-1, the expression of an MDR-1 promoter (-434/+147 fragment) - luciferase construct was unchanged or even inhibited by wt p53 that may be secondary to wt p53-mediated cytotoxicity. Thus, partial promoter constructs may not accurately represent endogenous MDR-1.

Recently it has been shown that the VHL tumor suppressor targets the hypoxia-inducible transcription factor (HIF-1) for ubiquitin-dependent degradation by the proteasome. Past mysteries of the p53 tumor suppressor help to solve the present puzzles of the VHL tumor suppressor. Thus, Mdm-2 targets the p53 tumor suppressor for ubiquitin-dependent degradation by the proteasome, but, in addition, the p53 transcription factor induces Mdm-2, thus, establishing a feedback loop. Hypoxia or DNA damage by abrogating binding of HIF-1 with VHL and p53 with Mdm-2, respectively, leads to stabilization and accumulation transcriptionally active HIF-1 and p53. More detailed analysis depicts the VHL/HIF-1 pair as the p53/mdm-2 pair that is turned upside down, suggesting that VHL may be a HIF-1-inducible gene of the feedback loop. The extended model proposes that an oncoprotein and a tumor suppressor due to transactivation coupled with feedback protein degradation might form functional pairs (Rb/E7, E2F/Rb, E2F/Mdm-2, catenin/APC, p27, cyclin D1, Rb/gankyrin), thus, predicting missing links.

To develop a new approach to the treatment of primitive neuroectodermal tumors we evaluated the effect of the HMG-CoA reductase inhibitor lovastatin on the Ewing's sarcoma cell line CHP-100. Lovastatin induced neural morphology and markers including neuron-specific enolase and neurofilament protein. The acquisition of neural morphology required new mRNA synthesis, and cDNA microarray analysis confirmed that lovastatin altered the program of gene expression. After morphologic differentiation the cells underwent rapidly progressive apoptosis. In normal development of neuronal progenitors, differentiation signals trigger p21WAF1 accumulation, RB hypophosphorylation, enhanced RB-E2F-1 association, and G1 arrest, and these events have been shown to protect from apoptosis. In contrast, in the Ewing's sarcoma cells lovastatin triggered differentiation without causing cell cycle arrest: p21WAF1 was not induced, RB remained hyperphosphorylated, and RB protein expression and RB-E2F-1 association were markedly downregulated, suggesting that loss of an RB-regulated G1 checkpoint promoted apoptosis. Consistent with this hypothesis, adenoviral p21WAF1 decreased DNA synthesis and partially protected from lovastatin-induced cytotoxicity. The data demonstrate a new model for examining the genetic regulation of cell fate in a neural progenitor tumor and suggest a new approach to the treatment of this neoplasm.

Hypoxia limits tumor growth but selects for higher metastatic potential. We tested the functional activity of hypoxia-inducible factor-1 (HIF-1) in prostate cell lines ranging from normal epithelial cells (PrEC), hormone-dependent LNCaP, hormone-independent DU145, PC-3 to highly metastatic PC-3M cancer cell lines. We found that HIF-1-stimulated transcription was the lowest in PrEC and LNCaP cells and the highest in PC-3M cells. The induction by hypoxia of the HIF-1 dependent genes Cap43 and GAPDH was the highest in the most aggressive PC-3M cancer cells. Because these advanced prostate cancer cell lines have lost p53 function, this further shifts a balance from p53 to HIF-1 transcriptional regulation, and a high ratio of HIF-1-dependent:p53-dependent transcription was a marker of the advanced malignant phenotype. Transient transfection of HIF-1alpha expression vector induced transcription from p21 promoter construct in prostate cancer cell lines. Furthermore, hypoxia slightly induced p21 mRNA in these cells. However, neither expression of p21 nor hypoxia caused growth arrest in PC-3M cells. Therefore, high inducibility of HIF-1-dependent genes, loss of p53 functions with high ratio of HIF-1-dependent:p53-dependent transcription, and loss of sensitivity to p21 inhibition is a part of hypoxic phenotype associated with aggressive cancer behavior.

Increasing the complexity of their models, p53s are stabilized either in order to function (wt p53) or due to the loss of function (mutant p53) with acquiring a mysterious prion-like ability to drive the normal p53 into the abnormal conformation to gain new functions. As already recognized, the loss of trans-activating function leads to a stabilization of mutant p53 because of the disappearance of the p53-inducible proteins, which otherwise directly (Mdm-2) or indirectly (p21) target p53 for degradation. Simplifying further, I will discuss that the loss of function results in a dominant-negative effect and gain-of-function (a dominant-positive effect). Thus, mutant p53 lacking trans-activation function nevertheless may retain the ability to repress transcription due to its competition with numerous transcription factors for their coactivators. When mutant p53 competes with wt p53, the inhibition of the wt p53-dependent transcription is perceived as a dominant-negative effect. Just like trans-repression, a dominant-negative effect requires an excess of p53 and, therefore, a 'dominant'-negative effect is not dominant. Furthermore, the stabilization of an endogenous mt p53 due to the loss of wt functions cannot occur in the presence of the wt p53 allele. Given the inability of mutant p53 to accumulate in the presence of wt p53, a dominant-negative effect does not naturally occur and, not surprisingly, heterozygous mt/wt cells are rare. The detection of a dominant-negative effect simply indicates that mutant p53 indeed has lost its function. Last, since mutant p53 loses some or most but not all activities and accumulates in the absence of wt allele, gain-of-function can be considered as an exaggeration of the remaining functions. Applications to cancer therapy are discussed. -Blagosklonny, M. V. p53 from complexity to simplicity: mutant p53 stabilization, gain-of-function, and dominant-negative effect.

Here we show that p53 protein is physically associated with tubulin in vivo and in vitro, and that it localizes to cellular microtubules. Treatment with vincristine or paclitaxel before DNA-damage or before leptomycin B treatment reduces nuclear accumulation of p53 and expression of mdm2 and p21. Overexpression of dynamitin or microinjection of anti-dynein antibody before DNA damage abrogates nuclear accumulation of p53. Our results indicate that transport of p53 along microtubules is dynein-dependent. The first 25 amino acids of p53 contain the residues that are essential for binding to microtubules. We propose that functional microtubules and the dynein motor protein participate in transport of p53 and facilitate its accumulation in the nucleus after DNA damage.

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., (Copyright 2000 Cancer Research Campaign.)

Lack of selectivity in the killing of tumor and normal cells is a major obstacle in cancer therapy. By inhibiting normal but not autonomous cell growth, we exploited the differences in cell cycle regulation to achieve a selective protection of nonautonomous cells against paclitaxel and other microtubule-active drugs. Tubulin polymerization, a primary effect of paclitaxel, can be dissociated from Bcl-2 phosphorylation and cytotoxicity in HL-60 cells. Growth arrest prevented paclitaxel-induced Bcl-2 phosphorylation and apoptosis without affecting paclitaxel-induced tubulin polymerization. We abrogated the effects of paclitaxel on MCF-10A immortalized breast cells, while preserving its effects on MCF-7 cancer cells. Unlike MCF-7 cells, MCF-10A cells were arrested by epidermal growth factor withdrawal, precluding paclitaxel-induced Bcl-2 phosphorylation. Furthermore, the inhibition of the epidermal growth factor receptor kinase with low doses of AG1478 arrested growth of MCF-10A but not MCF-7 cells. Pretreatment with AG1478 did not affect paclitaxel-induced Bcl-2/Raf-1 phosphorylation in MCF-7 but abrogated such phosphorylation in MCF-10A. Exploitation of growth factor dependency may allow the protection of normal cells from microtubule-active drugs.

Nickel (Ni2+) and cobalt (Co2+) mimic hypoxia and were used as a tool to study the role of oxygen sensing and signaling cascades in the regulation of hypoxia-inducible gene expression. These metals can produce oxidative stress; therefore, it was conceivable that reactive oxygen species (ROS) may trigger signaling pathways resulting in the activation of the hypoxia-inducible factor (HIF)-1 transcription factor and up-regulation of hypoxia-related genes. We found that the exposure of A549 cells to Co2+ or Ni2+ produced oxidative stress, and although Co2+ was a more potent producer of ROS than Ni2+, both metals equally increased the expression of Cap43, a hypoxia-regulated gene. The coadministration of hydrogen peroxide with metals induced more ROS; however, this did not further increase the expression of Cap43 mRNA. The free radical scavenger 2-mercaptoethanol completely suppressed ROS generation by CoCl2 and NiCl2 but did not diminish the induced Cap43 gene expression. The activity of the HIF-1 transcription factor as assessed in transient transfection assays was stimulated by Ni2+, hypoxia, and desferrioxamine, but this activation was not diminished when oxidative stress was attenuated nor was HIF-dependent transcription enhanced by hydrogen peroxide. We conclude that ROS are produced during the exposure of cells to metals that mimic hypoxia, but the formation of ROS was not involved in the activation of HIF-1-dependent genes.

The efficacy of anticancer therapy is limited by the development of drug resistance. While the role of p53 in the intrinsic sensitivity of human cancer cells to paclitaxel (PTX) remains controversial, its role in acquired paclitaxel resistance has never been addressed. In this study we examined the p53 status of three paclitaxel selected human ovarian carcinoma sublines, resistant to paclitaxel due to acquired beta-tubulin mutations which impair paclitaxel's interaction with tubulin. In contrast to parental cells which have wt p53, in all PTX-resistant sublines p53 was functionally inactive. Two of the resistant sublines expressed high levels of transcriptionally inactive p53 protein, each with a distinct point mutation in codons 236 and 239 of the DNA binding domain. The third subline presented a novel p53 pseudo-null phenotype as a result of markedly decreased wt p53 mRNA expression. Introduction of ectopic wt p53 had no effect on PTX sensitivity in both parental and resistant cells, while it induced p21WAF1/CIP1, demonstrating an intact p53 pathway. While PTX resistance is primarily conferred by the tubulin mutations, the loss of functional p53 observed in all clones, suggests that this loss may facilitate the development of resistance potentially by providing a clonal advantage which promotes the isolation of paclitaxel resistant cells.

Purpose: Once a relapse occurs following primary endocrine treatment, metastatic prostate cancer is one of the most therapy-resistant human neoplasms. Ketoconazole is used for complete androgen deprivation, and recent data suggest it has direct activity against prostate cancer cells., Materials and Methods: LNCaP, DU145, and PC3 cells, human prostate cancer cell lines, and HL60, a human leukemia cell line, were lysed and soluble proteins were harvested. Cells were plated in 96-well flat bottom plates and then exposed to the pharmacological agents, ketoconazole, vinblastine and paclitaxel. DNA synthesis was monitored by 3H-thymidine incorporation., Results: We demonstrate that ketoconazole exerts a cytostatic effect on a panel of human prostate cancer cell lines, with IC50 of 4 to 5 microg./ml., 12 microg./ml., and 25 microg./ml. for LNCaP, PC3/PC3M, and DU145 cells, respectively. On the other hand, using microtubule-active drugs, vinblastine and paclitaxel, we found that PC3M and PC3 cells were more resistant than either DU145 or LNCaP cells. This resistance was associated with a lesser degree of Raf-1 and Bcl-2 phosphorylation following exposure to microtubule-active drugs. Combinations of microtubule-active drugs with ketoconazole were a beneficial treatment in DU145 cancer cells. Furthermore, ketoconazole blocked recovery of all the prostate cancer cell lines following 24 hours-pulse treatment with vinblastine., Conclusion: Pulse-administration of vinblastine followed by continuous administration of ketoconazole warrants investigation in the treatment of hormone-independent metastatic prostate cancer.

Cell-cycle checkpoint mechanisms, including the p53- and p21-dependent G(2) arrest that follows DNA damage, are often lost during tumorigenesis. We have exploited the ability of DNA-damaging drugs to elicit this checkpoint, and we show here that such treatment allows microtubule drugs, which cause cell death secondary to mitotic arrest, to kill checkpoint-deficient tumor cells while sparing checkpoint-competent cells. Low doses of the DNA-damaging drug doxorubicin cause predominantly G(2) arrest without killing HCT116 cells that harbor wt p53. Doxorubicin treatment prevented mitotic arrest, Bcl-2 phosphorylation, and cell death caused by paclitaxel, epothilones, and vinblastine. In contrast, doxorubicin enhanced cytotoxicity of FR901228, an agent that does not affect microtubules. Low doses of doxorubicin did not arrest p21-deficient clones of HCT116 cells and did not protect these cells from cytotoxicity caused by microtubule drugs, but cells in which p21 expression was restored enjoyed partial protection under these conditions. Moreover, in p53-deficient clones of HCT116 cells doxorubicin did not induce either p53 or p21 and provided no protection against paclitaxel-induced cytotoxicity. Therefore, (a) p53-dependent p21 induction caused by doxorubicin protects from microtubule drug-induced cytotoxicity, and (b) pretreatment with cytostatic doses of DNA-damaging drugs before treatment with microtubule drugs results in selective cytotoxicity to cancer cells with defective p53/p21-dependent checkpoint.

Carcinogenic nickel compounds alter the program of gene expression in normal cells and induce a pattern of gene expression similar to that found in nickel-induced cancers. Here we have demonstrated that nickel exposure induced hypoxic signaling pathways by inducing hypoxia-inducible transcription factor-1 (HIF-1), which mediated the induction of genes required by cells to survive hypoxia. We also show that a new gene, Cap43, is dependent upon HIF-1 because only HIF-1-proficient cells induced Cap43 when exposed to either hypoxia or nickel. We also show that glyceraldehyde-3-phosphate dehydrogenase, a gene induced by hypoxia through HIF-1, was similar to Cap43 in that it required HIF-1-proficient cells to be induced by either nickel or hypoxia. These data demonstrate that nickel exposure turns on signaling for hypoxic stress, which may be important in its carcinogenesis.

BRCA1, a tumor suppressor protein implicated in hereditary forms of breast and ovarian cancer, is transcriptionally regulated in a proliferation-dependent manner. In this study, we demonstrate a substantial role for proteolysis in regulating the BRCA1 steady-state protein level in several cell lines. N-acetyl-leu-leu-norleucinal (ALLN), an inhibitor of the proteasome, calpain, and cathepsins, caused BRCA1 protein to accumulate in the nucleus of several human breast, prostate, and melanoma cell lines which express low or undetectable basal levels of BRCA1 protein, but not in cells with high basal expression of BRCA1. Protease inhibition did not increase BRCA1 synthesis, nor change its mRNA level, but it dramatically prolonged the protein's half-life. In contrast to ALLN, lactacystin and PS341, two specific proteasome inhibitors, as well as calpastatin peptide and PD150606, two selective calpain inhibitors, had no effect on BRCA1 stability, whereas ALLM, an effective calpain and cathepsin inhibitor but weak proteasome inhibitor, did stimulate accumulation of BRCA1. Moreover, three inhibitors of acidic cysteine proteases, chloroquine, ammonium chloride and bafilomycin, were as effective as ALLN. These results demonstrate that degradation by a cathepsin-like protease in fine balance with BRCA1 transcription is responsible for maintaining the low steady-state level of BRCA1 protein seen in many cancer cells.

Recent studies on paclitaxel (Taxol), a microtubule-stabilizing agent and effective anti-cancer drug, have identified numerous cellular and molecular effects, such as induction of cytokines and tumor-suppressor genes, indirect cytotoxicity due to secretion of tumor necrosis factor, vast activation of signal-transduction pathways and selective activity against cells lacking functional p53. Some of these results, including the immediate activation of signaling pathways and gene expression, have been observed only with paclitaxel concentrations 1,000-fold higher than those required for mitotic arrest and apoptosis. The effects of loss of p53 on paclitaxel cytotoxicity depend on cell type (normal murine fibroblasts vs. human cancer cells) and duration of exposure to paclitaxel; p53 status marginally affects paclitaxel sensitivity in human cancer. Although the biochemistry of mitosis and meiosis has been studied independently of research on the mechanism of action of anti-cancer drugs, it eventually provided insight into the effects of paclitaxel. For example, serine protein phosphorylation, which occurs during mitotic arrest or meiosis, explains paclitaxel-induced hyperphosphorylation of Bcl-2 and Bcl-xL. Although some observations are disputed, such mitotic arrest correlates with paclitaxel cytotoxicity, while there is currently no evidence that any paclitaxel effect at clinically relevant concentrations is independent of its tubulin-binding properties. Thus, paclitaxel exerts two types of effect: mitotic arrest with coincidental serine protein phosphorylation and cytotoxicity at clinically relevant concentrations as well as immediate activation of tyrosine kinase pathways and activation of gene expression at much higher concentrations., (Copyright 1999 Wiley-Liss, Inc.)

Micromolar concentrations of tamoxifen inhibit the activity of protein kinase C and were recently shown to inhibit prostate cancer cell growth in preclinical studies. Because micromolar concentrations can be attained with high-dose therapy, the clinical activity of high-dose tamoxifen was evaluated in patients with metastatic adenocarcinoma of the prostate. Between December 1993 and February 1997, 30 patients with hormone-refractory metastatic adenocarcinoma of the prostate were continuously administered tamoxifen at 160 mg/m2/day. Therapy was continued until disease progression. All study patients had failed prior treatment with combined androgen blockade, had castrate levels of testosterone, and were heavily pretreated, having received a median of three prior regimens. The average steady-state plasma concentration of tamoxifen was 2.96+/-1.32 microM (mean +/- SD). Grade 3 neurotoxicity was observed in 29% of patients and was rapidly reversible and readily managed with dose modification. Otherwise, grade 3 toxicities were rare. One partial response (80% decline in prostate-specific antigen) was observed (3.3%), whereas disease stabilization was observed in six patients (20%), for a combined partial response/stable disease response rate of 23%. Median time to progression was 2.1 months, and median survival time was 10.5 months. High-dose tamoxifen therapy was well tolerated and associated with micromolar concentrations of tamoxifen in human plasma, and it demonstrated activity, albeit limited, in a heavily pretreated patient cohort with hormone-refractory prostate cancer. These findings suggest that further investigation of the role of protein kinase C modulation in prostate cancer is warranted.