Interconversion of Cyclic Nucleotide-Activated and Cyclic Nucleotide-Independent Forms of a Protein Kinase from Beef Heart

A protein kinase activated by cyclic nucleotides was purified from beef heart. Upon exposure to adenosine 3':5'-cyclic monophosphate (cyclic AMP) during gel filtration on Sephadex G-200, the protein kinase dissociated into a cyclic nucleotide-independent protein kinase and a cyclic nucleotide-binding protein. A similar or identical cyclic nucleotide-independent protein kinase could be obtained in highly purified form by clution from a DEAE-cellulose column with 10(-6) M cyclic AMP; the cyclic AMP-binding protein was apparently retained by the resin. The addition of cyclic nucleotide-binding protein to cyclic nucleotide-independent protein kinase resulted in the reappearance of cyclic nucleotide-dependent protein kinase, which could be isolated by filtration on Sephadex G-200 in the absence of cyclic AMP. These results confirm and extend previous suggestions that cyclic nucleotides activate protein kinases by dissociating them from inhibitory, cyclic nucleotide-binding proteins.     

Treatment of advanced gastric carcinoma with mitomycin and doxorubicin

Thirty patients with advanced gastric carcinoma were treated with doxorubicin combined with mitomycin; only four (13%) responded. Myelosuppression was the major toxic effect; there was one toxic death. The overall median survival was 14 weeks (range, 2-42), which is no different from that reported for untreated patients. The combination of doxorubicin and mitomycin alone cannot be recommended for use in patients with advanced gastric carcinoma.     

Comparative cytotoxicity of folate-based inhibitors of thymidylate synthase and 5-fluorouracil ± leucovorin in MGH-U1 cells

Thymidylate synthase (TS) is a critical enzyme in the synthesis of DNA and an important target for cancer chemotherapy. 5-Fluorouracil (5FU) combined with leucovorin (LV) has been used to inhibit TS, and inhibition is dependent on the formation of a ternary complex between a folate cofactor, TS, and 5-fluorodeoxyuridine monophosphate (FdUMP), a metabolite of FU. The folate-based TS inhibitors CB3717, its analogs, and BW1843U89 have been synthesized as specific inhibitors of TS that do not require activation or the presence of a cofactor. We have compared the cytotoxicity of 5FU ± LV with that of these folate-based TS inhibitors in human bladder cancer MGH-U1 cells using a colony-forming assay. After a 6-h exposure, FU+LV, CB3717, dCB3717, or C2 methyl dideazafolate analogs demonstrated similar cytotoxic potency that was 0.96 to 2.9 times that of 5FU alone. A 24-h exposure did not increase the potency of 5FU+LV relative to 5FU alone, but there was a marked increase in the cytotoxicity of the dideazafolates as compared with 5FU+LV. Similarly, BW1843U89 was more cytotoxic than 5FU+LV. This was reflected in a 3.2- to 1333-fold decrease in the 50% inhibitory concentration (IC₅₀). Simultaneous exposure to LV and thymidine (TdR) protected MGH-U1 cells from the cytotoxicity of CB3717, its analogs, and BW1843U89. We conclude that (a) the folate-based TS inhibitors are more potent than 5FU+LV after a 24-h exposure, (b) protection by LV and TdR indicates that TS inhibition is the primary site of action, and (c) BW1843U89 is more potent than D1694 in MGH-U1 cells.This study was supported by a grant from the National Cancer Institute of Canada     

Resistance of MGH-U1 bladder cancer spheroids to vincristine.

We compared the cytotoxicity of vincristine in MGH-U1 human bladder cancer cells growing as exponential monolayer culture, spheroids and xenografts. Cells treated as spheroids were resistant to vincristine as determined by clonogenic survival and growth delay. The spheroid population had a smaller proportion of cells in G2 + M than monolayer cells. Cell derived from increasing depths of the spheroid viable rim had similar cell cycle distribution characteristics and sensitivity to vincristine. Prolonged treatment of spheroid did not increase vincristine cytotoxicity significantly. When cells derived from spheroids were treated as monolayers, the cytotoxicity was the same as that of cells maintained as monolayer cultures. The vincristine resistance observed in spheroids was also observed in xenografted tumours treated in vivo. Vincristine decreased the clonogenic survival of xenografted cells at in vivo doses which were greater than the LD10 for the mice. The in vitro cytotoxicity of the xenografted tumours at these lethal doses was similar to that of cells treated as spheroids. We conclude that vincristine resistance in spheroids may be attributed in part to the small proportion of cells traversing mitosis but not to the development of intrinsic resistance by passage through spheroid growth. Our results are consistent with cell cycle kinetics and limited penetration contributing to vincristine resistance in spheroids. The spheroid system can serve as a model of in vivo cytotoxicity for antineoplastic agents with cell cycle phase specificity such as vincristine.     

Physical activity and its impact on health outcomes. Paper 2: prevention of unhealthy weight gain and obesity by physical activity: an analysis of the evidence

The current guidelines for physical activity are based on the prevention of cardiovascular disease. In this article the magnitude and type of physical activity required to prevent unhealthy weight gain are assessed. Five categories of analyses are considered, ranging from the most rigorous analyses (based on D₂O¹⁸ measures of energy expenditure) to socio‐ecological associations. To standardize the approach, published work on the extent of exercise was expressed as a physical activity level (PAL), i.e. the ratio of total expenditure to the measured or estimated basal metabolic rate. D₂O¹⁸, direct monitoring and measurements of activity patterns and detailed prospective studies of substantial population groups all suggest that a PAL of ≥1.8 is required to limit the proportion of overweight and obese adult men. Data on women are more difficult to interpret because women are less active and the relationship with physical activity is usually less clear. Post‐obese women with a PAL of >1.75 do not regain weight and other data are consistent with the need for a PAL of ≥1.8. The analyses in both sexes are based predominantly on adults living in a Western society with the ready availability of energy‐dense foods. Vigorous activity is more clearly linked to weight stability, allows a higher intensity of exercise for general activities and shortens the time needed for achieving a PAL of 1.8. This activity level is equivalent to an additional 60–90 min of brisk walking in adults who normally undertake only modest exercise. These demands are greater than the current suggested levels for cardiovascular benefit and imply the need for different environmental policies, rather than health education policies, if societies are to become generally more active and avoid unhealthy weight gain.     

Phase II study of the farnesyl transferase inhibitor R115777 in patients with advanced non-small-cell lung cancer.

PURPOSE: This phase II study was undertaken to define the efficacy and pharmacodynamics of R115777, a farnesyl transferase inhibitor, in the first-line treatment of patients with advanced non-small-cell lung cancer. PATIENTS AND METHODS: Forty-four patients with measurable stage IIIB (pleural effusion) or stage IV disease received 193 courses of treatment (median, 2.0; range, 1 to 22) with R115777 300 mg administered orally twice daily for 21 of every 28 days. Buccal mucosa samples and peripheral blood mononuclear cells (PBMCs) were collected before and after 8 days of treatment to evaluate inhibition of farnesyl transferase in vivo. RESULTS: No objective complete or partial responses were documented. Seven patients (16%; 95% confidence interval [CI], 8% to 31%) had disease stabilization for greater than 6 months. Median survival was 7.7 months (95% CI, 6.5 to 10.5) and time to progression was 2.7 months (95% CI, 1.9 to 3.1). The most severe toxicity was neutropenia (9% grade 3, 7% grade 4) and the most common toxicities were anemia (50% grade 1 or 2, 5% grade 3) and anorexia (50% grade 1 or 2, 2% grade 3). Mild peripheral neuropathy occurred in 25% of patients. Evidence of farnesyl transferase inhibition was documented in 83% of patients. CONCLUSION: Single-agent R115777 was well tolerated in patients with advanced NSCLC, but demonstrated minimal clinical activity. Inhibition of farnesylation in vivo was consistently documented. On the basis of promising results of farnesyl transferase inhibitor combinations with standard chemotherapy agents, future studies of this agent in NSCLC should be in combination with systemic chemotherapy.