'Gain of function' phenotype of tumor-derived mutant p53 requires the oligomerization/nonsequence-specific nucleic acid-binding domain.

Tumor-derived p53 mutants can transcriptionally activate a number of promoters of genes involved in cellular proliferation. For this transactivation, mutant p53 does not use the wild-type p53 DNA-binding site, suggesting a mechanism of transactivation that is independent of direct DNA binding. Here we describe our analysis of the domain requirements for mutant p53 to transactivate promoters of the human epidermal growth factor receptor (EGFR), human multiple drug resistance 1 (MDR-1) and human proliferating cell nuclear antigen (PCNA) genes. We also report the identification of a structural domain required for the 'gain of function' property of mutant p53-281G. 'Gain of function' is measured as the tumorigenicity (in nude mice) of 10(3) murine cells expressing mutant p53 constitutively. We have generated internal deletion mutants of p53-281G deleting conserved domains I, II, III, IV and V, individually. We have also generated one deletion mutant eliminating amino acids 100 through 300 that removes four of the five conserved domains (II - V); another mutant, p53-281G del 393-327, deletes the oligomerization and nonsequence-specific nucleic acid-binding domains of p53. For the EGFR and MDR-1 promoters, all these mutants have significantly lower transactivation ability than intact p53-281G. These deletion mutants, however, significantly activated the pCNA promoter, suggesting that the mechanism of transactivation of the PCNA promoter is different from that of the EGFR and MDR-1 promoters. When expressed constitutively in 10(3) cells, p53-281G del 393-327 was found to be defective in inducing tumor formation in nude mice although intact p53-281G was very efficient. Thus, our results suggest that structural domains near the C-terminus are needed for 'gain of function'.

Intratumoral toremifene therapy and tissue distribution in the baboon.

The purpose of the present study was to evaluate the tissue distribution of toremifene (TOR) in baboons following intra-tissue injections and to examine the effectiveness of intratumoral TOR therapy of baboons with various spontaneous neoplasms. Five healthy baboons (Papio sp.) were used to examine the distribution of TOR following intra-tissue injections. Twenty-three different tissue specimens were collected for HPLC analysis. In addition, four baboons with various spontaneous neoplasms (myxoma, squamous cell carcinoma, lymphosarcoma and adenocarcinoma) were treated with intratumoral TOR and their responses were evaluated. Tissue TOR distribution was also examined in these animals. In the tissue distribution study, target tissue/serum TOR concentration ratios ranged from 138 to 8873 and the target tissue/other tissue ratios ranged from 1.2 to 2428. The distribution of TOR was very favorable, with the highest concentrations outside the injection sites noted in adjacent organs. A marked response was observed in the myxoma and partial responses were observed in the other three cases. Drug level analysis data from these four animals revealed tissue concentrations similar to those seen in the TOR tissue distribution study. Intratumoral administration of TOR can achieve effective local tumor and tissue concentrations, while systemic distribution via circulation to other organs is limited.

Flow cytometry: potential utility in monitoring drug effects in breast cancer.

Flow cytometric analysis of DNA ploidy and S-phase fraction are well recognized prognostic indicators in breast cancer. The present paper deals with the widening of the applications of flow cytometry to monitoring the effectiveness of antiestrogen therapy, detecting clonal selection and emergence of drug resistance, and monitoring chemosensitizing properties of drugs. Antiestrogen activity can be studied by DNA flow cytometry to address clinical research problems such as patient-specific pharmacokinetics, dosing compliance, and acquired antiestrogen resistance. Patient plasma specimens containing various concentrations of triphenylethylenes can be monitored for drug-induced effects using cell cycle measurements and correlated to in vivo drug levels. DNA flow cytometry has also been instrumental in the study of the effects of prolonged low-dose (0.5 microM for > 100 days) tamoxifen treatment on human estrogen receptor negative MDA-MB-231 cells, where it was shown that tamoxifen may significantly alter cell cycle kinetics and tumorigenicity of these cells, selecting a new, more aggressive, and rapidly growing clone. Lastly, it has been shown that the chemosensitizing properties of another triphenylethylene antiestrogen, toremifene, on estrogen receptor negative, multidrug resistant MDA-MB-231-A1 human breast cancer cells can be studied using flow cytometric analysis. Toremifene (and its metabolites N-desmethyltoremifene and toremifene IV) are able to "resensitize" MDA-MB-231-A1 cells to vinblastine and doxorubicin, as reflected in a marked shift of cells to G2/M phase of the cell cycle. Flow cytometry is a widely available technique that might be applied clinically to monitor, at the cellular level, drug effects on tumors, including the modulators of drug resistance.

Prolonged tamoxifen exposure selects a breast cancer cell clone that is stable in vitro and in vivo.

The effects of long-term tamoxifen exposure on cell growth and cell cycle kinetics were compared between oestrogen receptor (ER)-positive (MCF-7) and ER-negative (MDA-MB-231) cell lines. In the MCF-7 cell line, prolonged tamoxifen exposure (0.5 mumol/l for > 100 days) blocked cells in G0-G1 of the cell cycle, and slowed the doubling time of cells from 30 to 59 h. These effects corresponded to an increase in the cellular accumulation of tamoxifen over time [mean area under concentration curve (AUC) = 77.92 mumoles/10(6)/cells/day]. In contrast, in the MDA-MB-231 cell line, long-term tamoxifen exposure had no obvious effect on the doubling time, and reduced cellular tamoxifen accumulation (mean AUC = 50.50 mumoles/10(6)/cells/day) compared to the MCF-7 cells. Flow cytometric analysis of MDA-MB-231 cells demonstrated that a new tetraploid clone emerged following 56 days of tamoxifen exposure. Inoculation of the MDA-MB-231 tetraploid clone and MDA-MB-231 wildtype cells into the opposite flanks of athymic nude mice resulted in the rapid growth of tetraploid tumours. The tetraploid tumours maintained their ploidy following tamoxifen treatment for nine consecutive serial transplantations. Histological examination of the fifth transplant generation xenografts revealed that the tetraploid tumour had a 25-30 times greater mass, area of haemorrhage and necrosis, a slightly higher mitotic index and was more anaplastic than the control neoplasm. The control wildtype MDA-MB-231 tumours maintained a stable ploidy following tamoxifen treatment until the eighth and ninth transplantation, when a tetraploid population appeared, suggesting that tamoxifen treatment may select for this clone in vivo. These studies suggest that prolonged tamoxifen exposure may select for new, stable, fast growing cell clones in vitro as well as in vivo.

A breast cancer clone selected by tamoxifen has increased growth rate and reduced sensitivity to doxorubicin.

The in vivo growth rate and the chemosensitivity patterns of a cell clone selected by tamoxifen from the estrogen receptor-negative human breast cancer cell line MDA-MB-231 was studied in the nude mouse model and with flow cytometry. To investigate the growth rate of the wild-type and clone cells in vivo, the cells were inoculated into the opposite flanks of 5 male nude mice. Drug sensitivity to doxorubicin (10 ng/mL), vinblastine (1 ng/mL), and paclitaxel (1 ng/mL) was examined in wild-type/clone cell mixture using flow cytometry. Northern blot technique was used to study the expression of mdr-1 messenger RNA in both the wild-type and the clone cells. The tumors derived from the clone and wild-type cells were, following a 3-week growth period, 260.2 +/- 78.8 mm2 vs. 68.3 +/- 50.8 mm2 in size, respectively (P < 0.001). Following a 28-day continuous exposure, doxorubicin was selectively, toxic to the wild-type cells, while having no apparent effect on the clone population. However, paclitaxel- and vinblastine-treated wild-type/clone cell mixtures did not exhibit a differential cytotoxic effect on either cell population. It was concluded that the clone selected by tamoxifen shows an aggressive growth rate in vivo and an altered chemosensitivity pattern to doxorubicin in vitro.

Toremifene enhances cell cycle block and growth inhibition by vinblastine in multidrug resistant human breast cancer cells.

The clinical study of compounds that modulate multidrug resistance has been hindered by both the toxicities of these agents and the inability to monitor their effectiveness at the level of the tumor cell. Previously, toremifene has been shown to be well tolerated clinically and to sensitize multidrug resistant cells to the effects of cytotoxic chemotherapeutic agents. The chemosensitizing properties of toremifene in estrogen receptor negative, multidrug resistant MDA-MB-A1 human breast cancer cells were studied using flow cytometric analysis and growth inhibition assays. Cell cycle kinetics of MDA-MB-A1 cells were not significantly affected by treatment with either toremifene, N-desmethyltoremifene, Toremifene IV or vinblastine alone, as the majority of cells remained in G0/G1. However, preincubation with toremifene or one of its metabolites for 72 hours followed by treatment for one hour with vinblastine caused a marked shift of cells to G2/M, as cells appeared to be blocked in that phase of the cell cycle. This result was nearly identical to the effect of vinblastine alone on vinblastine-sensitive MDA-MB-231 breast cancer cells and can be interpreted as a "resensitization" by toremifene of MDA-MB-A1 cells to vinblastine. This chemosensitizing effect of toremifene was accompanied by an enhanced inhibition of cell growth by vinblastine. The chemosensitizing effects of toremifene or one of its metabolites in combination with cytotoxic chemotherapy can be effectively monitored by flow cytometry, an easily accessible technique.