James K. V. Willson

Heterogeneity of human colon carcinoma.

SummaryIn order to better understand colon cancer, a model system reflecting the heterogenous nature of this disease was developed and used in the development of new cytotoxic and non-cytotoxic therapeutic approaches. A large bank of colon carcinoma cell lines was established from primary human colon carcinomas and grouped based on their tumorigenicity in athymic mice, their growth rates in soft agarose and in tissue culture, and their secreted levels of carcinoembryonic antigen. These cell lines were later characterized based on cell surface proteins and antigens detected with antisera raised against a differentiated colon carcinoma cell line. Although these biochemical markers correlated with the biological classification of these cell lines, there was still extensive heterogeneity within each group in all properties examined. This colon carcinoma cell system was used to study natural vs. selected resistance to the anticancer drug mitomycin C (MMC). The differing IC50 values in vitro were reflected in the inhibition by MMC of xenograft growth in athymic mice. A new, more readily bioactivatable analogue of MMC was tried and shown to be more active in vitro and in vivo, suggesting that rapid efflux of the drug before activation may be important in examining causes of resistance to MMC. Another approach to the treatment of colon cancer is the use of non-cytotoxic agents such as growth factors and differentiation agents to restore normal growth to the malignant cells. We have isolated and characterized two types of polypeptides from colon carcinoma cells and conditioned medium from these cells. The first, transforming growth factors (TGFs) confer a transformed phenotype on non-transformed fibroblasts while the second, tumor inhibitory factors (TIFs), inhibits the anchorage independent growth of transformed cells. The fact that extracts of colon carcinoma cells contain both activities suggests that the heterogeneity of the cell lines could be due to different levels of TGFs and TIFs produced. The effectiveness of differentiation agents to restore normal growth control using a transformed mouse embryo cell line was examined. Treatment of these cells with differentiation agents restored normal growth control to these cells. An increased synthesis of TGFs resulted from these treatments. Therefore, differentiation agents may be useful in non-cytotoxic treatment. The use of this model system for human colon carcinoma will hopefully lead to more effective drugs for the treatment of colon cancer in man.

A novel mechanism of resistance to epidermal growth factor receptor antagonism in vivo

Epidermal growth factor receptor (EGFR) is widely expressed in a number of solid tumors including colorectal cancers. Overexpression of this receptor is one means by which a cell can achieve positive signals for survival and proliferation; another effective means is by constitutive activation of EGFR. We have elucidated the role of constitutive EGFR signaling in malignant progression by stably transfecting colon cancer cells with a human transforming growth factor-alpha cDNA (a ligand for EGFR) under repressible control by tetracycline. We show that constitutive expression of transforming growth factor-alpha and its subsequent constitutive activation of EGFR allows for cancer cell survival in response to environmental stress in vitro and in vivo as well. The reversal of constitutive EGFR activation results in the loss of downstream mitogen-activated protein kinase and Akt activation, and a reduction in xenograft size that is associated with decreased proliferation and increased apoptosis. We used CI-1033, a small molecule antagonist of EGFR, to dissect an activation pathway that shows the ability of ERBb2 to activate Akt, but not Erk in the face of EGFR antagonism. This novel escape mechanism is a possible explanation of why anti-EGFR therapies have shown disappointing results in clinical trials.

Augmentation of methotrexate cytotoxicity in human colon cancer cells achieved through inhibition of thymidine salvage by dipyridamole

In HCT 116 cells, a human colon cancer cell line, the levels of thymidine (0.6 microM) and hypoxanthine (9 microM) contributed to the tissue culture medium by the fetal bovine serum significantly reduced the growth inhibition and lethality produced by 0.1 microM methotrexate. Dipyridamole, an inhibitor of nucleoside transport, potentiated the growth inhibitory effects of methotrexate when the cells were grown in medium that was changed daily. However, when the medium was supplemented with dialyzed serum, methotrexate cytotoxicity was not increased by dipyridamole. Similarly, in cloning experiments, dipyridamole increased the cell killing produced by methotrexate. The potentiation of methotrexate toxicity produced by dipyridamole was mediated through inhibition of thymidine uptake. The uptake of 1 microM thymidine was inhibited 50% by 0.12 microM dipyridamole but neither hypoxanthine nor guanine uptake was decreased by dipyridamole (5 microM). As a result, the decrease in dTTP pools produced by methotrexate was augmented by dipyridamole. In contrast, dipyridamole did not influence the effect of methotrexate on ribonucleoside triphosphate pools. HCT 116 cells avidly salvaged low concentrations of thymidine, and methotrexate increased this capacity. Conversion of 0.11 microM thymidine to thymidine triphosphate was increased by 55%, from 16.6 to 25.7 pmoles/10(6) cells, following exposure to 1.0 microM methotrexate. Dipyridamole blocked this pool expansion. This study suggests that the salvage of physiological levels of thymidine may diminish the cytotoxic effects of methotrexate on human colon cancer cells. Inhibition of thymidine uptake by dipyridamole may be an effective strategy to increase the cytotoxicity of methotrexate.

Platinum Analogue Combination Chemotherapy: Cisplatin and Carboplatin-A Phase I Trial With Pharmacokinetic Assessment of the Effect of Cisplatin Administration on Carboplatin Excretion

Cisplatin (NSC 119875) and carboplatin (NSC 241240) are platinum (II) analogues with very different spectra of toxicity. Cisplatin dose is limited by nausea and vomiting, renal dysfunction, and dose-related peripheral neuropathy, whereas carboplatin is myelosuppressive. There are also clinical and laboratory data that suggest that these drugs may not be completely cross-resistant. Therefore, the following phase I trial of combination therapy with cisplatin and carboplatin was undertaken. Since carboplatin toxicity is enhanced in the presence of renal impairment, carboplatin excretion was also evaluated in selected patients at the maximum tolerated dose. Thirty-three patients received 50 mg/m2 cisplatin and doses of carboplatin between 160 mg/m2 and 400 mg/m2. Sequential 20-minute infusions of carboplatin and then cisplatin were able to be administered at the standard doses of carboplatin (320 and 400 mg/m2) with thrombocytopenia to the degree expected if carboplatin alone had been given. However, 280 mg/m2 carboplatin followed by 25 mg/m2 cisplatin/d X 3 caused unexpectedly severe thrombocytopenia in seven of eight patients (median platelet nadir 45,000/microL; range, 12 to 321,000/microL; nadir was less than 90,000 in seven of eight patients). In three patients treated with 280 mg/m2 carboplatin plus 25 mg/m2/d X 3 cisplatin, pharmacokinetics of carboplatin were compared during consecutive monthly cycles without and with cisplatin. Modestly increased areas under the curve (AUC) for carboplatin (15% and 35%) with cisplatin were seen in the two patients who experienced more pronounced platelet suppression with combination therapy. No other limiting or unusual toxicity was seen with this combination. Responses, primarily in platinum responsive tumors, were seen. The combination of cisplatin plus carboplatin is feasible and merits further study.

The Development of Techniques That Permit the Selection and Growth of Malignant Cells from Human Colonic Carcinomas

Publisher Summary This chapter discusses the development of techniques that permit the selection and growth of malignant cells from human colonic carcinomas. The technique which, was developed involved a human colon cancer cell line bank utilizing culture methodologies that permit successful establishment of cell lines from the majority of cancer specimens cultured. Cell lines have been established from neoplastic specimens which include the full spectrum of the clinical disease including an adenoma, localized and aggressive primary cancers, and metastases. These cell lines display biologic and pharmacologic diversity, making the VACO cell line bank an attractive disease-specific model for use in drug discovery programs as well as studies to understand the biologic progression of colon cancer. It was recognized that cell lines undergo selective pressures during in vitro cultivation and as a result may not retain all phenotypes present in the individual clinical tissue of origin. On the other hand, the preservation of such diverse characteristics in these established lines suggests that they exhibit at the least a portion of the diversity present in the patients, and they represent a valuable and practical model system for the laboratory investigation of colon cancer.