Loss of normal p53 function confers sensitization to Taxol by increasing G2/M arrest and apoptosis.

The anticancer agent paclitaxel (Taxol) stabilizes tubulin polymerization resulting in arrest in mitosis and apoptotic cell death. Normal human fibroblasts depleted of functional p53 by SV40 T antigen or HPV-16 E6, and primary embryo fibroblasts from p53 null mice showed seven- to ninefold increased cytotoxicity by paclitaxel. Reduced levels of p53 correlated with increased G2/M phase arrest, micronucleation, and p53-independent paclitaxel-induced apoptosis. Surviving cells with intact p53 progressed through mitosis and transiently accumulated in the subsequent G1 phase, coincident with increased p53 and p21cip1,waf1 protein levels. These results are in contrast to studies linking p53 loss with resistance to DNA damaging anticancer agents.

Paclitaxel inhibits progression of mitotic cells to G1 phase by interference with spindle formation without affecting other microtubule functions during anaphase and telephase.

Very low concentrations of paclitaxel, a clinically active anticancer agent isolated from the bark of the Pacific yew tree, were found to produce micronuclei in human colon carcinoma cells, suggesting inhibition of mitotic spindle assembly or function. The possibility that paclitaxel acts at the level of the mitotic spindle was investigated by evaluating its ability to inhibit the progression of mitotic cells to G1 phase. Paclitaxel inhibited mitotic progression with a median inhibitory concentration of 4 nM, a concentration equivalent to the median cytotoxic concentration, without arresting cells in mitosis. A direct correlation was shown to exist between the cytotoxic potency and ability to inhibit mitotic progression for analogues of paclitaxel and antimicrotubule agents but not for the topoisomerase II-active agents etoposide and teniposide. After release from the nocodazole block, cells synchronized in mitosis remained sensitive to very low concentrations of paclitaxel for < 30 min, the time required for spindle formation, yet remained sensitive to vinblastine for > 90 min. This result indicates that very low concentrations of paclitaxel inhibit formation of mitotic spindles in cells without affecting function of preformed spindles and without arresting cells in mitosis. Continuous exposure to low nanomolar concentrations of paclitaxel for more than one cell cycle resulted in cells with DNA contents > 4C and as much as 8C. These results support a hypothesis, that, by not being capable of segregating sister chromatids, paclitaxel-treated cells eventually reform nuclear membranes around individual or clusters of chromosomes, revert to G1 phase cells containing 4C DNA, and enter S phase, resulting in cells with as much as 8C DNA content. It is proposed that this is the primary cytotoxic mechanism of paclitaxel.

The multidrug resistance phenotype: 31P nuclear magnetic resonance characterization and 2-deoxyglucose toxicity.

In order to identify changes in 31P nuclear magnetic resonance (NMR) spectra associated with multiple drug resistance (MDR), a number of wild type and drug-resistant cancer cell lines were studied. The resistant cells included cells selected with various drugs, mainly Adriamycin, as well as cells transfected with the human multidrug resistance gene (MDR1 gene), which encodes P-glycoprotein. In most cases, 31P NMR spectra were significantly different from those of parental, drug-sensitive lines. The spectra of resistant cells generally indicated increased levels of ATP and phosphocreatine in the cytoplasm. These changes are compatible with the increased glucose utilization rate previously described for resistant cells. Major changes were also observed in the levels of glycerophosphocholine and glycerophosphoethanolamine. Changes in cellular metabolism reflected by 31P NMR spectra depend on the drug used to select the cells for MDR. The direction of these changes was not consistent for all cell lines studied and could not be directly attributed to expression of P-glycoprotein, suggesting that the changes may be related to alterations in metabolism and membrane function associated with other mechanisms of MDR. The results demonstrate the suitability of 31P NMR for studies of biochemical changes associated with MDR. The toxicity of 2-deoxyglucose, a glucose antimetabolite, was investigated in addition to the NMR studies and was found to be consistently higher in multidrug-resistant cells than in the parental drug-sensitive lines. For MCF-7 breast cancer cells, where several sublines with different levels of resistance were available, the toxicity was highest for the most resistant lines.

Isolation of amplified and overexpressed DNA sequences from adriamycin-resistant human breast cancer cells.

An MCF-7 human breast cancer cell line was selected which was 200-fold more resistant to Adriamycin than the wild type cell line. This Adriamycin-resistant (AdrR) cell line exhibited a multidrug-resistant phenotype and was cross-resistant to a wide range of antineoplastic agents including Vinca alkaloids, anthracyclines, and epipodophyllotoxins. Cytogenetic analysis of the AdrR cell line showed the presence of homogeneously staining regions on several chromosomes which were not present in the parental cell line. Using the technique of in-gel renaturation, DNA sequences which were amplified 50- to 100-fold in the AdrR cell line and which covered a total of over 140 kilobases were isolated. In addition, AdrR cells were found to contain amplified and overexpressed sequences which were homologous to hamster P-glycoprotein gene sequences. A hamster cDNA P-glycoprotein gene probe was used to screen a lambda gt10 cDNA library made from human AdrR cell line mRNA and human cDNA sequences homologous to the P-glycoprotein gene were isolated. Hybridization studies with the cloned human cDNA (pADR1) showed that the AdrR MCF-7 cell line contained a 60-fold amplification of this DNA sequence and that polyadenylated mRNA from the AdrR cell line contained a 4.8-kilobase transcript which was overexpressed 45-fold. There was a direct correlation between DNA and RNA copy number of this sequence and level of resistance among several MCF-7 Adriamycin-resistant cell lines. In situ hybridization studies demonstrated that the human P-glycoprotein gene sequence was found on chromosome 7q21.1 in normal human lymphocytes and that amplified DNA sequences isolated from the AdrR MCF-7 cells by the in-gel hybridization technique were linked to the human P-glycoprotein sequences in the homogeneously staining regions in the AdrR cells.

Eleutherobin, a novel cytotoxic agent that induces tubulin polymerization, is similar to paclitaxel (Taxol).

Eleutherobin is a novel natural product isolated from a marine soft coral that is extremely potent for inducing tubulin polymerization in vitro and is cytotoxic for cancer cells with an IC50 similar to that of paclitaxel. This compound is cross-resistant along with other multidrug-resistant agents against P-glycoprotein-expressing cells and is cross-resistant with paclitaxel against a cell line that has altered tubulin. In mechanistic studies, eleutherobin shares with paclitaxel the ability to induce tubulin polymerization in vitro and is most likely cytotoxic by virtue of this mechanism. Human colon carcinoma cells exposed to eleutherobin contain multiple micronuclei and microtubule bundles, and they arrest in mitosis, depending on concentration, cell line, and length of exposure. These morphological abnormalities appearing in cultured cells are indistinguishable from those induced by paclitaxel. Electron microscopy reveals that eleutherobin induces homogeneous populations of long, rigid microtubules similar to those formed by paclitaxel. Thus, eleutherobin is a new chemotype with a mechanism of action similar to that of paclitaxel and, as such, has promising potential as a new anticancer agent.

Preclinical antitumor activity of BMS-214662, a highly apoptotic and novel farnesyltransferase inhibitor.

BMS-214662 is a potent and selective inhibitor of farnesyltransferase (FTI). In rodent fibroblasts transformed by oncogenes, BMS-214662 reversed the H-Ras-transformed phenotype but not that of K-Ras or other oncogenes. In soft agar growth assays, BMS-214662 showed good potency in inhibiting H-ras-transformed rodent cells, A2780 human ovarian carcinoma tumor cells, and HCT-116 human colon carcinoma tumor cells. Inhibition of H-Ras processing in HCT-116 human colon tumor cells was more rapid than in H-Ras-transformed rodent fibroblast tumors. BMS-214662 is the most potent apoptotic FTI known and demonstrated broad spectrum yet robust cell-selective cytotoxic activity against a panel of cell lines with diverse histology. The presence of a mutant ras oncogene was not a prerequisite for sensitivity. Athymic and conventional mice were implanted s.c. with different histological types of human and murine tumors, respectively. BMS-214662 was administered both parenterally and p.o. and was active by all these routes. Curative responses were observed in mice bearing staged human tumor xenografts including HCT-116 and HT-29 colon, MiaPaCa pancreatic, Calu-1 lung, and EJ-1 bladder carcinomas. A subline of HCT-116, HCT-116/VM46, resistant to many standard cytotoxic agents by means of a multiple drug resistance mechanism, remained quite susceptible to BMS-214662, and borderline activity was achieved against N-87 human gastric carcinoma. Two murine tumors, Lewis lung carcinoma and M5076 sarcoma, were insensitive to the FTI. In a study performed using Calu-1 tumor-bearing mice, no obvious schedule dependency of BMS-214662 was observed. The FTI, BMS-214662, demonstrated broad spectrum activity against human tumors, but murine tumors were not as sensitive.

Expression of anionic glutathione-S-transferase and P-glycoprotein genes in human tissues and tumors.

The development of multidrug resistance in MCF-7 human breast cancer cells and the acquisition of broad resistance to xenobiotics in rat hyperplastic nodules are both associated with increased P-glycoprotein (mdr) gene expression as well as changes in activities of intracellular detoxication enzymes; among these changes is a significant increase in the activity of the anionic isozyme of glutathione-S-transferase (GST). We have isolated a cDNA encoding the human anionic glutathione-S-transferase, GST pi-1, from a cDNA library constructed from multidrug-resistant MCF-7 cells. The deduced amino acid sequence of GST pi-1 shows that while the human anionic GST displays 85% nucleotide and amino acid sequence homology to the rat anionic isozyme, it is markedly less related to human basic GST isozymes. We have examined the expression of GST pi and P-glycoprotein in 170 specimens of human tissues and tumors. P-Glycoprotein RNA expression was positive in eight of 23 lymphomas and two of 12 colon tumors; however, many other normal and malignant tissues, including lung, bladder, and breast tumors, had low or undetectable levels of P-glycoprotein RNA expression. In contrast, GST pi was readily detected in a wide variety of normal and malignant tissues. The level of GST pi mRNA expression in normal tissues was heterogeneous, with lowest levels found in liver and the highest levels found in lung, esophagus, and placenta. GST pi was also variably expressed in human tumors, with the lowest relative levels occurring in lymphoma and breast cancer and the highest levels found in lung cancer and head and neck tumors. In addition, comparison of paired specimens from the same patient indicated that GST pi expression was increased in many tumors relative to matched normal tissue.

Alprazolam vs amitriptyline in depressions with reduced REM latencies.

This study was designed to compare the antidepressant effects of alprazolam, a triazolobenzodiazepine, with amitriptyline hydrochloride in a group of patients with nonpsychotic, major depressions diagnosed by Research Diagnostic Criteria. A mean rapid eye movement latency of less than 65 minutes was required to enter this study. Dexamethasone suppression tests were conducted before treatment. By strictly applied Research Diagnostic Criteria, 83.6% of the subjects were endogenous, and 34.7% were inpatients. A significantly greater percentage of alprazolam-treated patients responded within the first seven days of treatment. By the end of this six-week trial, alprazolam was associated with significant reductions in Hamilton, Beck, Covi, Raskin, and Carroll Rating scores (pretreatment to posttreatment). However, by the end of treatment the effects of amitriptyline exceeded those of alprazolam on both the Hamilton and Beck scales. These data indicate that alprazolam is not as effective as amitriptyline in major depressions with a shortened rapid eye movement latency.

Preclinical pharmacology of BMS-275183, an orally active taxane.

BMS-275183 is a taxane, the mechanism of action of which is like other known taxanes, and is the polymerization of tubulin. BMS-275183 given p.o. was as effective as i.v. paclitaxel in five tumor models [murine M109 lung and C3H mammary 16/C, and human A2780 ovarian (grown in mice and rats) and HCT/pk colon]. It was active in one other tumor model (human HCT-116 colon) but inferior to parenteral paclitaxel. BMS-275183 given p.o. was active in a human, hormone-dependent, prostate tumor model, CWR-22, and just as effective as anti-androgen chemotherapy. In a schedule dependency study, increasing the interval of time between oral administrations resulted in greater cumulative dose tolerance and improved therapeutic outcome. Oral BMS-275183 was evaluated as a combination therapy in conjunction with i.v. paclitaxel. Therapeutic advantages were evident for tumor-bearing mice that received the oral taxane either after induction chemotherapy or between courses of such treatment. BMS-275183 is currently in Phase I clinical trials at multiple sites.

BMS-247550: a novel epothilone analog with a mode of action similar to paclitaxel but possessing superior antitumor efficacy.

BMS-247550, a novel epothilone derivative, is being developed by Bristol-Myers Squibb Company (BMS) as an anticancer agent for the treatment of patients with malignant tumors. BMS-247550 is a semisynthetic analogue of the natural product epothilone B and has a mode of action analogous to that of paclitaxel (i.e., microtubule stabilization). In vitro, it is twice as potent as paclitaxel in inducing tubulin polymerization. Like paclitaxel, BMS-247550 is a highly potent cytotoxic agent capable of killing cancer cells at low nanomolar concentrations. Importantly, BMS-247550 retains its antineoplastic activity against human cancers that are naturally insensitive to paclitaxel or that have developed resistance to paclitaxel, both in vitro and in vivo. Tumors for which BMS-247550 demonstrated significant antitumor activity encompass both paclitaxel-sensitive and -refractory categories, i.e., (a) paclitaxel-resistant: HCT116/VM46 colorectal (multidrug resistant), Pat-21 breast and Pat-7 ovarian carcinoma (clinical isolates; mechanisms of resistance not fully known), and A2780Tax ovarian carcinoma (tubulin mutation); (b) paclitaxel-insensitive: Pat-26 human pancreatic carcinoma (clinical isolate) and M5076 murine fibrosarcoma; and (c) paclitaxel sensitive: A2780 ovarian, LS174T, and HCT116 human colon carcinoma. In addition, BMS-247550 is p.o. efficacious against preclinical human tumor xenografts grown in immunocompromised mice or rats. Schedule optimization studies indicate that BMS-247550 is efficacious when administered frequently (every 2 days x 5) or intermittently (every 4 days x 3 or every 8 days x 2). These efficacy data demonstrate that BMS-247550 has the potential to surpass Taxol in both clinical efficacy and ease of use (i.e., less frequent treatment schedule and/or oral administration).

Role of renin classification for diuretic treatment of black hypertensive patients.

Previous studies in white and mixed-race hypertensive patient populations have generally found patients with low renin activity more responsive to diuretic therapy than patients with normal renin activity. Twenty-nine black patients (26 women and three men) with placebo diastolic blood pressure of 90 to 115 mm Hg were treated with spironolactone (100 to 400 mg/day) and hydrochlorothiazide (100/mg/day). Renin status was categorized by (1) the intravenous furosemide test, (2) ambulation during placebo, and (3) ambulation during spironolactone and hydrochlorothiazide treatment. Only seven patients were categorized identically with all methods. No method identified a low renin subgroup that was more responsive to either spironolactone or hydrochlorothiazide. Diastolic blood pressure fall with hydrochlorothiazide (18 mm Hg) and 400 mg/day of spironolactone (15 mm Hg) was similar. Thus, since black women with both low and normal renin activity are quite responsive to diuretics, renin classification to guide initial antihypertensive selection is not warranted.

Further evaluation of saline infusion for the diagnosis of primary aldosteronism.

Normal subjects, normal-renin hypertensive patients, and low-renin hypertensive patients were evaluated by intravenous saline infusion and with a fludrocortisone acetate (Florinef) protocol to clarify diagnostic criteria for primary aldosteronism that are recommended for the saline infusion protocol. The patients consumed a 200 mEq sodium, 70 mEq potassium diet for 6 days, and on the last 3 days received Florinef 0.5 mg orally twice daily. On Days 3 and 6, urinary aldosterone and tetrahydroaldosterone excretions were determined, and on Days 4 and 7 plasma aldosterone (PA) was determined at 0600 after overnight recumbency and at 0800 after 2 hours of walking. Although the level of normal PA suppression by saline infusion has been commonly defined as 10 ng/dl, a value of 5 ng/dl was originally recommended. In 20 normal subjects and 45 normal-renin hypertensive patients, we found that the PA was almost always suppressed below 5 ng/dl. In 18 of 75 low-renin patients including five with aldosterone-producing adenoma (APA), the PA was never suppressed below 10 ng/dl; thus, these 18 patients had classical primary aldosteronism by generally accepted criteria. The Florinef protocol was performed in eight of these 18 patients and was abnormal in all. An abnormal Florinef protocol was also found in seven of 15 patients studied with PA suppression after saline infusion to between 5 and 10 ng/dl, but in only one of 24 patients studied with PA suppression below 5 ng/dl. Additional studies in the subgroup with abnormal results from the Florinef protocol indicated that none of these patients had evidence of APA, so they had nontumorous primary aldosteronism (NTPA).(ABSTRACT TRUNCATED AT 250 WORDS)

Free triiodothyronine (T3) and thyroxine (T4) in a group of unipolar depressed patients and normal subjects.

We examined levels of free triiodothyronine (fT3) and free thyroxine (fT4) in serum from a group of 32 patients with unipolar major depression and 46 normal control subjects using the Amerlex (Amersham, Arlington Heights, IL) RIA procedures for these hormones. Free T3 levels were significantly lower (p less than 0.004) in the depressed patients as a group (5.02 +/- 1.01 pmol/L, mean +/- SD) than in the normal control subjects (5.74 +/- 1.23 pmol/L). Free T3 levels were lower (p less than 0.01) in depressed men (5.25 +/- 1.43 pmol/L) when compared with male control subjects (6.46 +/- 1.01 pmol/L). Depressed women (4.78 +/- 0.60 pmol/L) also had lower T3 levels than did the female control subjects (5.09 +/- 1.06 pmol/L), but the difference was not statistically significant. Lower fT3 levels were also observed in melancholic depressed patients when compared with nonmelancholic depressed patients or when compared with normal control subjects. No differences in fT4 were observed between groups in this study.

Dexamethasone response, thyrotropin-releasing hormone stimulation, rapid eye movement latency, and subtypes of depression.

Most prior studies of mood disorders have used a single laboratory test to assist in differential diagnosis, prediction of treatment response, and prediction of relapse. This study compared three laboratory measures in a combined in- and outpatient sample of depressed patients. Dexamethasone suppression test (DST) nonsuppression occurred in 46% of patients with endogenous major depression, in 15% with nonendogenous major depression, and in 56% with bipolar, depressed phase disorder. A blunted thyrotropin-releasing hormone stimulation test (TRH-ST) occurred in 25% of patients with endogenous, 10% with nonendogenous, and 44% with bipolar, depressed phase disorder. Reduced REM latency was found in 65% of endogenous major depressions, in 34% of nonendogenous major depressions, and in 53% of bipolar, depressed phase disorders. Fifty-one percent of those with reduced REM latency also evidenced DST nonsuppression. When the endogenous major depression and bipolar, depressed phase groups were combined, 28% had no laboratory abnormality, whereas 8% evidenced all three. These findings suggest that 1) endogenous/nonendogenous unipolar groups are distinguished by all three laboratory tests; 2) most patients with a blunted TRH-ST also evidence DST nonsuppression; and 3) one half of patients with reduced REM latency evidence DST nonsuppression. Sensitivity is greatest and specificity is lowest for REM latency, followed by the DST and then the TRH-ST.

Keynote address: multidrug resistance: a pleiotropic response to cytotoxic drugs.

Tumor cells exposed in tissue culture to one of several different classes of antineoplastic agents, including anthracyclines, vinca alkaloids, epipodophyllotoxins, and certain antitumor antibiotics, can develop resistance to the selecting agent and cross resistance to the other classes of agents. This phenomena of multidrug resistance is generally associated with decreased drug accumulation and overexpression of a membrane glycoprotein. This membrane protein, referred to as P-glycoprotein, apparently acts as an energy-dependent drug efflux pump. Multidrug resistance in human MCF-7 breast cancer cells selected for resistance to adriamycin (AdrR MCF-7) is associated with amplification and overexpression of the mdr1 gene which encodes P-glycoprotein. A number of other changes are also seen in this resistant cell line including alterations in Phase I and Phase II drug metabolizing enzymes. Similar biochemical changes occur in a rat model for hepatocellular carcinogenesis and are associated in that system with broad spectrum resistance to hepatotoxins. The similar changes in these two models of resistance suggests that these changes might be part of a battery of genes whose expression can be altered in response to cytotoxic stress, thus rendering the cell resistant to a wide variety of cytotoxic agents.

Synthesis and biological evaluation of novel C-4 aziridine-bearing paclitaxel (taxol) analogs.

Three novel C-4 aziridine-bearing paclitaxel analogs, 3-5, have been synthesized during the course of our continuing effort at C-4 modification. The key step in the synthesis is the aziridine ring formation at the C-4 position via an intramolecular Mitsunobu reaction. The syntheses and the biological evaluation of these C-4 aziridine-containing derivatives are herein discussed.

Naamidine A is an antagonist of the epidermal growth factor receptor and an in vivo active antitumor agent.

The known 2-aminoimidazole alkaloid naamidine A (1) was isolated from a Fijian Leucetta sp. sponge as an inhibitor of the epidermal growth factor (EGF) receptor. The compound exhibited potent ability to inhibit the EGF signaling pathway and is more specific for the EGF-mediated mitogenic response than for the insulin-mediated mitogenic response. Evaluation in an A431 xenograft tumor model in athymic mice indicated that naamidine A exhibited at least 85% growth inhibition at the maximal tolerated dose of 25 mg/kg. Preliminary mechanism of action studies indicate that the alkaloid fails to inhibit the binding of EGF to the receptor and has no effect on the catalytic activity of purified c-src tyrosine kinase.

Synthesis and biological evaluation of 2-acyl analogues of paclitaxel (Taxol).

The anticancer drug paclitaxel (Taxol) has been converted to a large number of 2-debenzoyl-2-aroyl derivatives by three different methods. The bioactivities of the resulting analogues were determined in both tubulin polymerization and cytotoxicity assays, and several analogues with enhanced activity as compared with paclitaxel were discovered. Correlation of cytotoxicity in three cell lines with tubulin polymerization activity showed reasonable agreement. Among the cell lines examined, the closest correlation with antitubulin activity was observed with a human ovarian carcinoma cell line.

Synthesis and antitumor activity of novel C-7 paclitaxel ethers: discovery of BMS-184476.

The preparation of C-7 paclitaxel ethers is described. Various substituted ethers were prepared via activation of the corresponding methylthiomethyl ether followed by alcohol addition. Variation of the C-7 ether group as well the 3' side chain position led to the discovery of a novel taxane, BMS-184476 (4), with preclinical antitumor activity superior to paclitaxel.

Concurrent unilateral chromatid damage and DNA strand breakage in response to 6-thioguanine treatment.

The delayed cytotoxicity of 6-thioguanine (TG) may relate to the arrest of cells in G2 upon completion of one cell cycle after drug exposure. In Chinese hamster ovary (CHO) cells, both the unilateral chromatid damage in G2 chromosomes, determined by induction of premature condensed chromosome condensation [Maybaum and Mandel, Cancer Res. 43, 3852 (1983)], and incorporation of TG into DNA resulting in DNA strand breakage [Christie et al., Cancer Res. 44, 3665 (1984)] were correlated with cytotoxicity. We have studied the correlation between strand breakage and unilateral chromatid damage in L1210 cells. DNA breaks were detected only when cells were treated with TG (0.25 microM) for one cell cycle time (12 hr) followed by 12 hr in drug-free medium containing [3H]thymidine (TdR) to label the DNA. After simultaneous incubation of cells with drug and label during the first or second 12-hr period, strand breaks were not found. Strand breaks increased with dose, which correlated with greater cytotoxicity (0.01 to 0.25 microM). Treatment of cells with 0.25 microM TG for 12 hr, and transfer to drug-free medium for 12 hr prior to making prematurely condensed chromosomes (PCC), resulted in unilateral chromatid damage. Prominent curving of G2 chromosomes with gapping and diffuse staining of one of the sister chromatids occurred. The 4-fold increase in the percentage of cells in G2 compared with control cells suggested G2 arrest. When cells were treated with TG for 12 hr and PCC made immediately, neither the arrest of cells in G2 nor unilateral chromatid damage was observed. These data suggest that strand breaks and unilateral chromatid damage occur in the second cell cycle after TG exposure and that this damage may be important in TG-delayed cytotoxicity.