Patrick G. Johnston

Regulation of Thymidylate Synthase in Human Colon Cancer Cells Treated with 5-Fluorouracil and Interferon-Gamma

The effects of fluorouracil (5-FU) and interferon-gamma (IFN-gamma) on the regulation of thymidylate synthase (TS) gene expression were investigated in the human colon cancer H630 cell line. By Western immunoblot analysis, TS protein levels in H630 cells were increased 3-, 5.5-, 5-, and 2.5-fold after 8-, 16-, 24-, and 36-hr exposure to 1 microM 5-FU, respectively. When H630 cells were exposed to varying concentrations of 5-FU (0.3-10 microM) for 24 hr, increases in TS protein up to 5.5-fold were observed. A 24-hr exposure to 1 microM 5-FU resulted in a 4.5-fold increase in the level of TS protein, whereas in 5-FU/IFN-gamma-treated cells TS protein was increased by only 1.8-fold, compared with control cells. IFN-gamma treatment alone did not affect TS protein levels, relative to control. Northern blot analysis revealed no changes in TS mRNA levels when H630 cells were exposed either to 1 microM 5-FU for 8-36 hr, to varying concentrations of 5-FU (0.3-10 microM) for 24 hr, or to the combination of 5-FU and IFN-gamma. Pulse-labeling studies with [35S]methionine demonstrated a 3.5-fold increase in net synthesis of TS in cells treated with 1 microM 5-FU, whereas the level of newly synthesized TS increased only 1.5-fold in cells treated with 5-FU/IFN-gamma, compared with control cells. Pulse-chase studies revealed that the half-lives of TS protein in control and 5-FU-treated cells were equivalent. These findings demonstrate that the increase in TS protein after 5-FU exposure and the subsequent inhibitory effect of IFN-gamma on TS protein expression are both regulated at the post-transcriptional level.

Prognostic importance of thymidylate synthase expression in early breast cancer.

PURPOSE To assess the prognostic importance of thymidylate synthase (TS) expression in breast tumors of patients with early-stage breast cancer, and to determine whether the benefit of chemotherapy (CT) is associated with TS expression. PATIENTS AND METHODS The level of TS expression was evaluated in 210 node-negative and 278 node-positive patients enrolled onto Trial V of the International Breast Cancer Study Group ([IBCSG] formerly the Ludwig Breast Cancer Study Group) with a median follow-up time of 8.5 years. TS expression was assessed using the immunohistochemical method with the monoclonal antibody TS 106 on paraffin-embedded tissue specimens. RESULTS High TS expression was associated with a significantly worse prognosis in node-positive but not in node-negative breast cancer patients. Twenty-seven percent of node-positive patients with high TS expression were disease-free at 10 years, compared with 44% of node-positive patients with low TS expression (P = .03). Forty-one percent of patients with node-positive high-TS-expressing tumors were alive after 10 years, compared with 49% of those with low TS expression (P = .06). The association between TS and disease-free survival (DFS) and overall survival (OS) was independent of other prognostic factors such as tumor size, tumor grade, nodal status, vessel invasion, estrogen receptor (ER)/ progestin receptor (PR) status, c-erb B-2, or Ki-67 expression. In node-positive patients, six cycles of standard adjuvant cyclophosphamide, methotrexate, and fluorouracil ([5-FU] CMF) CT improved DFS and OS compared with one cycle of perioperative CMF therapy. The magnitude of this benefit was greatest in patients whose tumors had high TS expression (P < .01 for DFS; P < .01 for OS). Node-negative patients demonstrated no difference in outcome to CT based on TS expression; however, the power to detect differences was limited by the small number of events in this group. CONCLUSION In early-stage breast cancer, high TS expression is associated with a significantly worse prognosis in node-positive patients. Node-positive patients with high TS levels demonstrate the most significant improvement in DFS and OS when treated with six cycles of conventional adjuvant CMF therapy.

Stch encodes the 'ATPase core' of a microsomal stress 70 protein.

The stress70 protein chaperone family plays a central role in the processing of cytosolic and secretory proteins. We have cloned a human cDNA, designated Stch, that is conserved in rat tissues and which encodes a novel microsome‐associated member of the stress70 protein chaperone family. Stch mRNA is constitutively expressed in all human cell types and is induced by incubation with the calcium ionophore A23187, but not by exposure to heat shock. Inspection of the predicted amino acid sequence reveals that the STCH product contains a unique hydrophobic leader sequence and shares homology within the amino terminal domains of the stress70 gene family, but has a 50 residue insertion within the ATP‐binding domains and truncates the carboxyl terminal peptide‐binding region. Immunofluorescent and subcellular analyses show that STCH migrates predominantly as a 60 kDa species and is enriched in a membrane‐bound microsome fraction. In contrast to purified BiP and dnaK, however, STCH demonstrates ATPase activity that is independent of peptide stimulation. Stch, therefore, encodes a calcium‐inducible, microsome‐associated ATPase activity with properties similar to a proteolytically cleaved N‐terminal HSC70/BiP fragment. This truncated stress70 molecule may allow increased diversity in cellular responses to protein processing requirements.

The cellular interaction of 5-fluorouracil and cisplatin in a human colon carcinoma cell line

The combination of 5-fluorouracil (5-FU) and cisplatin (CDDP) has been shown to have synergistic cytotoxicity in human tumours, but the biochemical mechanism for this interaction remains unclear. Therefore, the aim of this study was to investigate the interaction of 5-FU and CDDP in a human colon carcinoma cell line, NCI H548. A 24 h exposure to 5-FU resulted in a 5-FU IC50 value of 24.2 +/- 4.5 microM, Dm 22.6 microM; exposure to CDDP for 2 h resulted in a IC50 value of 20.8 +/- 8.0 microM, Dm 21.9 microM. When cells were exposed simultaneously to 5-FU for 24 h and CDDP for the initial 2 h, or when cells were treated with CDDP for 2 h followed by various concentrations of 5-FU for 24 h, no greater than additive cytotoxicity was observed. In contrast, when cells were treated with 5-FU for 24 h prior to CDDP for 2 h, a greater than additive interaction was noted (5-FU IC50 1.2 +/- 0.6 microM, Dm 1.3 microM, CI 0.45). Thymidine 10 microM partially reversed the growth inhibitory effects of the 5-FU/ CDDP combination. Using both immunological and biochemical assays, no notable differences in the total amount of TS enzyme or the fraction of bound TS enzyme could be detected in the absence or presence of CDDP. No notable differences could be detected in intracellular reduced folate pools, FdUMP or FUTP pools, or 5-FU incorporation into RNA or DNA with the addition of CDDP to 5-FU. DNA fragmentation studies revealed that the combination of 5-FU followed by CDDP demonstrated a greater degree of single-stranded DNA fragments in parental (P = 0.024) and newly synthesised DNA (P = 0.025) compared with the administration of CDDP prior to 5-FU or either drug alone. The increase in smaller DNA fragment size was reversed with the addition of thymidine (10 microM). These findings suggest that the interaction of 5-FU and CDDP is associated with a greater degree of fragmentation of both nascent and parental DNA.

New Concepts for the Development and Use of Antifolates

Approximately one‐third of all cases of colorectal carcinoma present in an advanced and, therefore, incurable stage. For these patients, the development of new chemotherapeutic strategies is of central importance. Biochemical modulation of 5‐fluorouracil (5‐FU) has resulted in approximately a two‐fold increase in activity of 5‐FU. Recent preclinical investigations suggest that interferon can also modulate the activity of 5‐FU and may result in enhanced response rates in patients. One of the critical mechanisms of resistance to 5‐FU appears to be the acute induction in thymidylate synthase (TS) levels following therapy with inhibitors of this enzyme. This mechanism is based on a novel autoregulatory feedback pathway wherein the TS protein regulates its own translational efficiency. Regulatory function of the enzyme is dependent on its state of occupancy by either the physiologic ligands or inhibitors, including fluoropyrimidines and antifolates. Ongoing efforts are directed towards utilizing knowledge of this protein/messenger RNA interaction for therapeutic benefit. Given the importance of TS, our laboratory has developed antibodies capable of quantitating the levels of this enzyme in fresh or paraffin‐embedded tissues. Preliminary investigations suggest that the level of TS has prognostic importance in patients with rectal carcinoma and may be used to predict responsiveness to fluoropyrimidine agents. Novel strategies utilizing dual modulation of 5‐FU with leucovorin and interferon are under investigation in both the advanced and adjuvant disease settings. Emerging mechanistic concepts regarding TS, along with the development of new, more potent inhibitors will hopefully result in future therapeutic gains.

Fazarabine: A report of response in a patient with multiply relapsed embryonal cell carcinoma

This report describes a patient with multiply relapsed and advanced metastatic embryonal cell carcinoma who achieved a pathological partial remission (PR) following treatment with a Phase I chemotherapeutic agent, arabinosyl-5-azacytosine (Ara-AC), followed by orchiectomy. The antimetabolic Ara-AC is worthy of further study in patients with germ-cell tumors. It would appear that the antimetabolite Ara-AC may have significant activity in patients with germ-cell tumors.