Cancer Chemotherapeutic Regimens Research Articles

Aldehyde Dehydrogenase-Mediated Cellular Relative Insensitivity to the Oxazaphosphorines1

As judged by findings in preclinical models, determinants of cellular sensitivity to cyclophosphamide and other oxazaphosphorines include two cytosolic aldehyde dehydrogenases, viz., ALDHlAl and ALDH3Al. Each catalyzes the detoxification of the oxazaphosphorines; thus, cellular sensitivity to these agenh decreases as cellular levels of ALDHlAl and/or ALDH3Al increase. Of particular clinical relevance may be that stable sublines, relatively insensitive to the oxazaphusphorines Jue to elevated ALDHlAl or ALDH3Al levels, emerged when cultured human tumor cells were exposed only once to a high concentration of one of these agents for 30 to 60 minutes. Whether differences in cellular levels of either enzyme accounts for the clinically-encountered uneven therapeutic effectiveness of the oxazaphosphorines remains to be determined. However, it has already been established that measurable levels of these enzymes are found in some, but not all, tumor types, and that in those tumor types where measurable levels are present, e.g., infiltrating ductal carcinomas of the breast, they vary widely from patient to patient. Potentially useful clinical strategies that might be pursued if it turns out that ALDHIAl and/or ALDH3Al are, indeed, clinically operative determinants of cellular sensitivity to the oxazaphosphorines include 1) individualizing cancer chemotherapeutic regimens based, at least in part, on the levels of these enzymes in the malignancy of interest, and 2) sensitizing tumor cells that express relatively large amounts of ALDHlAl and/or ALDH3A 1 to the oxazaphosphorines by preventing the synthesis of these enzymes, e.g., with antisense RNA, or by introducing an agent that directly inhibits the catalytic action of the operative enzyme. Further, the fact that ALDHlAl and ALDH3Al are determinants of cellular sensitivity to the oxazaphosphorines provides the rationale for the investigation of two additional strategies with clinical potential, viz., decreasing the sensitivity of vulnerable and essential normal cells, e.g., pluripotent hematopoietic cells, to the oxazaphosphorines by selectively transferring into them the genetic information that encodes 1) ALDHlAl or ALDH3Al, or 2) a signaling factor, the presence of which would directly or indirectly, stably upregulate the expression of these enzymes.

3′-azido-3′-deoxythymidine cytotoxicity and metabolism in the human colon tumor cell line HCT-8

We have reported that 3′-azido-3′-deoxythymidine (AZT) possesses significant cytotoxicity in human tumor models when combined with agents that inhibit de novo thymidylate (dTMP) synthesis, such as 5-fluorouracil (FUra) and methotrexate (MTX). To aid in the further development of these and related cancer chemotherapeutic regimens, this study was undertaken to identify the biochemical processes relevant to the induction of AZT cytotoxicity in the model human colon tumor cell line HCT-8. The ic 50 of AZT in this cell line after a 5-day exposure was 55 μM. In cells incubated for 5 days with various concentrations of [ 3H]AZT alone, both [ 3H]AZT nucleotide pools and [ 3H]AZT incorporation into DNA increased as the concentration of AZT in the medium increased. In addition, a 5-day exposure to AZT, at medium concentrations ⩽ 100 μM, resulted in a reduction in dTMP synthase (EC 2.1.1.45; methylene tetrahydrofolate:deoxyuridine-5′-monophosphate C methyltransferase) and dThd kinase (EC 2.7.1.27; ATP: thymidine phosphotransferase) activities, compared with cells incubated without drug. The ic 50 of AZT was unchanged when the medium concentration of dThd was increased from 0.1 to 50 μM. Increasing the concentration of dThd to 50 μM also did not affect intracellular pools of [ 3H]AZTDP and [ 3H]AZTTP or the degree to which [ 3H]AZT was incorporated into cellular DNA, but did reduce intracellular [ 3H]AZTMP by ∼ 75%. The degree to which 3′-amino-3′-deoxythymidine (AMT) was generated from AZT and incorporated into DNA also was not affected by varying the medium concentration of dThd. However, the amount of [ 3H]-AMT detected in DNA, ⩽ 3 pmol/10 6 cells at medium concentrations of [ 3H]AZT ⩽ 100 μM, was below that associated with significant cytotoxicity in these cells. These data support the notion that, in this model, AZT cytotoxicity is determined by the relative size of AZTTP pools and its utilization in DNA synthesis. Studies to verify this relationship assessed the effect of alterations in the concentration of dTTP and [ 3H]AZTTP on [ 3H]AZT incorporation into newly synthesized DNA in vitro, using DNA polymerases isolated from HCT-8 cells. The results of these studies confirmed that alterations in the concentration of either dTTP or AZTTP to reduce the dTTP/AZTTP ratio resulted in an increase in AZT incorporation into DNA. These findings are discussed in light of their biochemical implications and relevance to ongoing clinical trials.

Circadian rhythm of cellular proliferation in the human rectal mucosa

Circadian rhythms of DNA synthesis and cellular proliferation in the gastrointestinal mucosa have been well documented in animal models. This investigation was designed to determine whether similar rhythms could be demonstrated in the human rectal epithelium: 24 studies were performed in 16 healthy men under fasting (n = 14) and fed (n = 10) conditions. Rectal mucosal biopsy specimens were obtained through a proctoscope every 2 or 3 hours for a 24-hour span. Ex vivo measurements of tritiated thymidine incorporation into DNA were made on the mucosal samples. Feeding and time of day were each found to have an effect on the rate of thymidine incorporation into the DNA of rectal mucosal cells. Both fasted and fed subjects showed significant circadian rhythms in thymidine incorporation, which peaked at about 7 am. Fasting lowered the overall mean thymidine uptake without altering the rhythm. Thymidine uptake generally reflects the amount of DNA synthesis in the sampled tissue. Therefore, these data may be important in the design of cancer chemotherapeutic regimens that use drugs specifically active during DNA synthesis.