pyrazofurin and Breast-Neoplasms

The purpose of this phase II clinical trial was to evaluate the effectiveness of triazinate and pyrazofurin in the treatment of advanced breast cancer. Although an occasional patient experienced stability of disease, neither agent, as employed in this study, was able to produce an objective antitumor effect in this group of patients with previous chemotherapeutic exposure. At the dosages used in this study, neither agent can be recommended for further treatment of advanced breast cancer.

Topics: Adult; Aged; Amides; Antibiotics, Antineoplastic; Breast Neoplasms; Drug Administration Schedule; Drug Evaluation; Drug Therapy, Combination; Female; Folic Acid Antagonists; Humans; Infusions, Parenteral; Middle Aged; Pyrazoles; Ribonucleosides; Ribose; Triazines

Abnormalities in the p53 tumor suppressor gene have been shown to affect cellular processes related to cell cycle control and gene amplification. In this study we compare the status and function of wild-type p53 in MCF-7 breast cancer cells with sublines selected for resistance to chemotherapeutic agents having different mechanisms of action. Sublines that were resistant to melphalan, pyrazafurin, mitoxantrone, etoposide and PALA all retained expression of wild-type p53. Methotrexate-resistant MCF-7 cells were unusual heterozygotes that expressed a wild-type and dominant, in-frame p53 deletion mutant and the doxorubicin-resistant cells expressed only mutant p53. Analysis of the G1 checkpoint after treatment with ionizing radiation revealed that the pyrazafurin-, melphalan- and mitoxantrone-resistant cells arrested strongly in G1. The etoposide- and PALA-resistant cells had an intermediate G1 arrest phenotype and the methotrexate- and doxorubicin-resistant cells had a minimal G1 arrest phenotype. mRNA and protein analyses of downstream effector genes, including P21CIP1/Waf1, mdm2, Gadd 45 and the retinoblastoma protein, did not entirely differentiate sublines having a strong versus intermediate G1 arrest phenotype. Neither the p53 status nor the strength of the G1 arrest could be correlated with cell survival after ionizing radiation. When drug-sensitive MCF-7 cells were treated with the same chemotherapeutic agents, p53 and p21CIP1/Waf1 levels increased between 2- and 14-fold. Together these data suggest that other cellular factors likely play a role in overcoming the inhibitory effects of ionizing radiation on p53 in drug-resistant breast cancer cells.

Topics: Amides; Antineoplastic Agents; Aspartic Acid; Blotting, Western; Breast Neoplasms; Cell Cycle; Clone Cells; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Drug Resistance, Neoplasm; Enzyme Inhibitors; Etoposide; Female; Flow Cytometry; Gene Expression; Genes, p53; Humans; Immunohistochemistry; Melphalan; Mitoxantrone; Neoplasm Proteins; Nuclear Proteins; Phosphonoacetic Acid; Polymerase Chain Reaction; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Pyrazoles; Ribonucleosides; Ribose; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Cultured wild-type MCF-7 human breast cancer cells and two MCF-7 sublines that overproduce enzymes of the de novo pyrimidine biosynthetic pathway were compared with regard to: rate of de novo biosynthesis of uracil nucleotides, sensitivity of the de novo and salvage pathways to the concentration of intracellular uracil nucleotides, and potential of exogenous uridine at concentrations equivalent to plasma levels to affect de novo pyrimidine biosynthesis. The PALAR MCF-7 subline, which is resistant to N-(phosphonacetyl)-L-aspartate and has 5.2 times the activity of the first de novo enzyme as the wild-type MCF-7 cells, synthesizes uracil nucleotides via the de novo pathway at a rate that is 5.8 times that of the wild type MCF-7 cells. The PYRR MCF-7 subline, which is resistant to pyrazofurin and has 15.1 times the activity of orotate phosphoribosyltransferase as the wild-type MCF-7 cells, synthesizes uracil nucleotides via the de novo pathway at a rate that is 1.4 times that of wild-type MCF-7 cells. These results are consistent with carbamyl phosphate synthetase being the rate-controlling step of de novo pyrimidine biosynthesis. In the presence of exogenous uridine at concentrations equivalent to that found in plasma (4.4-8.6 microM), the uracil nucleotide pool of wild-type MCF-7 cells was expanded by 20% and de novo synthesis was inhibited by 55%. Incubation of PALAR MCF-7 cells with uridine at concentrations between 7.3 and 16.8 microM caused a 40% increase in the uracil nucleotide pool and a 30% inhibition of de novo synthesis. De novo synthesis of uracil nucleotides in PYRR MCF-7 cells was not affected by a greater than 10-fold increase in the uracil nucleotide pool. Salvage of [14C] uridine was inhibited by an expanded uracil nucleotide pool in the wild-type and PYRR MCF-7 cells but was not inhibited in the PALAR MCF-7 cell line. These results demonstrate that, although the overproduced enzymes exhibit substrate affinities and specificities in cell-free preparations similar to those of the wild-type enzymes, in intact cells the resistant cell lines exhibit marked differences in the control of de novo and salvage pyrimidine biosynthetic pathways by intracellular uracil nucleotides.

Topics: Amides; Aspartate Carbamoyltransferase; Aspartic Acid; Breast Neoplasms; Carbamoyl-Phosphate Synthase (Ammonia); Cell Line; Dihydroorotase; Drug Resistance; Female; Humans; Orotate Phosphoribosyltransferase; Orotidine-5'-Phosphate Decarboxylase; Phosphonoacetic Acid; Pyrazoles; Pyrimidines; Ribonucleosides; Ribose; Uracil Nucleotides; Uridine