Higher levels of thymidylate synthase gene expression are observed in pulmonary as compared with hepatic metastases of colorectal adenocarcinoma.

PURPOSE: It has been observed previously that the pulmonary metastases of colorectal adenocarcinoma are less responsive to therapy with fluorouracil (FUra) as compared with other sites of metastasis (liver, local). To investigate the basis of this chemoresistance, the levels of thymidylate synthase (TS) mRNA and protein were measured, as TS expression has been shown to be predictive of response to therapy in colorectal cancer. MATERIALS AND METHODS: Tumors were obtained from 19 patients with metastatic colorectal cancer (12 hepatic and seven pulmonary). TS expression was measured by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and TS protein levels were measured by Western blotting. The presence of TS amplification was assessed by Southern blotting. Levels of p53 protein were determined using immunohistochemistry. RESULTS: TS mRNA expression was shown to be significantly higher in the pulmonary metastases (mean TS/beta-actin ratio, 19.7; n = 7) as compared with the hepatic metastases (mean TS/beta-actin ratio, 4.7; n = 11) of colorectal cancer. Lower TS expression was observed in patients with hepatic metastases who had received prior FUra versus patients who had not been treated. High levels of TS expression in some samples was associated with low-level (two to three gene copies) increases in TS gene copy numbers and this was observed more frequently in the pulmonary metastatic samples. The increased gene copy numbers occurred both in samples with wild-type p53 and those with mutant p53 tumor-suppressor gene as determined by immunohistochemistry. CONCLUSION: High levels of TS enzyme may be the basis of the lack of response of pulmonary metastases to FUra treatment.

Increased methotrexate polyglutamylation in acute megakaryocytic leukemia (M7) compared to other subtypes of acute myelocytic leukemia.

Acute myelocytic leukemia (AML) is a malignancy that is intrinsically resistant to methotrexate (MTX). AML blasts, when incubated with radiolabeled MTX, form lower amounts of long chain polyglutamates compared to acute lymphocytic leukemia (ALL) blasts, thus providing an explanation for their lack of responsiveness to MTX. Leukemic blasts obtained from two children with acute megakaryocytic leukemia (M7 subtype) when incubated with radiolabeled MTX showed increased accumulation of total as well as long chain MTX polyglutamates, comparable to levels previously demonstrated in another subtype of AML, acute monocytic leukemia (M5), as well as in blasts from patients with pre-B ALL. We suggest that M7-AML patients with blasts showing increased MTX polyglutamylation might benefit from treatment with MTX.

Pretreatment of colon carcinoma cells with Tomudex enhances 5-fluorouracil cytotoxicity.

The cytotoxic effect of sequence and dose of Tomudex (TX) and 5-fluorouracil (FUra) on an HCT-8 colon carcinoma cell line using a clonogenic assay was evaluated. Synergistic cell kill was obtained with 24 h of exposure to TX followed by 4 h of exposure to FUra. Marginal synergy was obtained with the same sequence but with a 5-day exposure to FUra. The reverse sequence, FUra (either 4 h or 5 days), followed by TX (24 h), resulted in less-than-additive cell kill. The synergistic effect was not due to augmented inhibition of thymidylate synthase, as determined by the measurement of thymidylate synthase activity by tritium release from [5-3H]2'-deoxyuridine. Surprisingly, an increase in intracellular levels of phosphoribosylpyrophosphate was observed after 24 h of exposure to TX, suggesting the possibility of an indirect effect of TX and/or its polyglutamates on purine biosynthesis. Moreover, we observed an increased formation of FUra nucleotides in the cells preexposed to TX, likely due to the increased intracellular levels of phosphoribosylpyrophosphate, that as a consequence led to an enhanced incorporation of FUra into RNA and increased cell killing.

Characterization and drug sensitivity of four newly established colon adenocarcinoma cell lines to antifolate inhibitors of thymidylate synthase.

Four new cell lines were established from the primary tumors of patients with untreated colorectal adenocarcinoma. Drug sensitivity and characterization of these cell lines was performed. Three of the four cell lines formed colonies in soft agar and all were tumorigenic in nude mice. The cell lines were morphologically similar but had differences in growth characteristics. Two of the cell lines, C18 (CCCL-4) and C29 (CCCL-6), had a longer doubling time compared with C85 (CCCL-1) and C86 (CCCL-2). The C18 and C29 cell lines had chromosome 17 abnormalities and evidence by immunohistochemistry of a mutant p53 and had decreased levels of thymidylate synthase and dihydrofolate reductase proteins, associated with decreased thymidylate synthase catalytic activity in C18 and no detectable activity in C29. Raltitrexed and GW1843U89 showed potent cytotoxic activity and all four cell lines displayed similar cytotoxicity to these folate thymidylate synthase inhibitors. The C18 and C29 cell lines were in general resistant to the other agents tested (methotrexate, 5-fluorouracil, nolatrexed) when compared with the C85 and C86 cell lines. These new cell lines may be useful for the study of colorectal adenocarcinoma and for evaluating new drugs or treatment schedules.

gamma-Glutamyl hydrolase and folylpolyglutamate synthetase activities predict polyglutamylation of methotrexate in acute leukemias.

Decreased methotrexate (MTX) long-chain polyglutamate formation is associated with MTX resistance whereas high levels of MTX polyglutamate accumulation are found in the blasts of leukemia patients who respond to therapy and have improved outcome. The steady-state level of long-chain MTX polyglutamates depends on the balance of activities of two enzymes: folylpolyglutamate synthetase (FPGS), which adds glutamates to MTX in a gamma-carboxyl linkage, and gamma-glutamyl hydrolase (GGH) or conjugase, which sequentially removes the terminal glutamate residue of MTX polyglutamates. FPGS and GGH activities as well as the formation of total and long-chain MTX polyglutamates were measured after incubation with [3H]MTX in 15 blast samples from patients with acute leukemias (myeloid and lymphoid). The ratio between GGH and FPGS activities was better at predicting the amount of polyglutamate accumulated in the 24-h [3H]MTX assay compared to the determination of either activity alone. The linear regression curve relating the relative levels of long-chain polyglutamates/total polyglutamates with the ratio of GGH/FPGS showed an r value of 0.81 (P < 0.001). These data suggest that the evaluation of both these enzymes at diagnosis may be used as a predictor of MTX polyglutamylation and therefore for response to MTX therapy and outcome.

All-trans-retinoic-acid- and growth-factor- mediated induction of alkaline phosphatase activity in freshly isolated chronic myeloid leukemia cells.

Reduced or absent neutrophil alkaline phosphatase (NAP) activity is a common feature of neutrophilic granulocytes from patients with chronic myeloid leukemia (CML). In this study we examined whether NAP activity could be restored in vitro by stimulating CML cells with different promoters such as all-trans-retinoic acid (ATRA), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF). The results obtained indicated that ATRA and G-CSF, either alone or in combination, were effective in inducing NAP activity in CML cells, whereas GM-CSF was not. Further, NAP restoration in ATRA- and G-CSF-treated cultures was accompanied by increased morphologic differentiation of the CML clone. It might be concluded that the CML clone could be driven in vitro by ATRA and G-CSF both to achieve granulocytic maturation and to correct functional NAP-related defects.

Disparate affinities of antifolates for folylpolyglutamate synthetase from human leukemia cells.

Previous work showed that acute myelocytic leukemia blasts accumulate less long chain polyglutamates of methotrexate (MTX) than acute lymphocytic leukemia blasts when incubated with this radiolabeled antifolate. This difference likely explains the increased sensitivity of lymphoid leukemias to short-term exposure of MTX as compared with myeloid leukemias. In this study, we examined the basis for differences between long chain MTX polyglutamate accumulation between different leukemia cell types using both leukemia cell lines and blasts freshly isolated from blood of leukemic patients. The major difference found between leukemia cells that accumulate long chain polyglutamates and those that do not were differences in Km values for the enzyme folylpolyglutamate synthetase. Km values did not change with partial purification of this enzyme, indicating that interfering substances in crude lysates were not responsible for this difference. We postulate that there may be differences in the properties of this enzyme related to tissue specific expression. In contrast to MTX, both Tomudex (Zeneca Pharmaceuticals, Wilmington, DE) and 1843U89, potent inhibitors of thymidylate synthetase, have low Kms for folylpolyglutamate synthetase, and polyglutamate forms of these inhibitors are accumulated to the same degree in both myeloid and lymphoid acute leukemia cells, paralleling the equivalent cytotoxicity found between myeloid and lymphoid leukemia cell lines. Based on these results, we believe a clinical trial of Tomudex in patients with acute myeloid leukemia is warranted.