Five-chlorodeoxycytidine, a tumor-selective enzyme-driven radiosensitizer, effectively controls five advanced human tumors in nude mice.

PURPOSE: The study's goals were as follows: (1) to extend our past findings with rodent tumors to human tumors in nude mice, (2) to determine if the drug protocol could be simplified so that only CldC and one modulator, tetrahydrouridine (H4U), would be sufficient to obtain efficacy, (3) to determine the levels of deoxycytidine kinase and dCMP deaminase in human tumors, compared to adjacent normal tissue, and (4) to determine the effect of CldC on normal tissue radiation damage to the cervical spinal cord of nude mice. METHODS AND MATERIALS: The five human tumors used were as follows: prostate tumors, PC-3 and H-1579; glioblastoma, SF-295; breast tumor, GI-101; and lung tumor, H-165. The duration of treatment was 3-5 weeks, with drugs administered on Days 1-4 and radiation on Days 3-5 of each week. The biomodulators of CldC were N-(Phosphonacetyl)-L-aspartate (PALA), an inhibitor of aspartyl transcarbamoylase, 5-fluorodeoxycytidine (FdC), resulting in tumor-directed inhibition of thymidylate synthetase, and H4U, an inhibitor of cytidine deaminase. The total dose of focused irradiation of the tumors was usually 45 Gy in 12 fractions. RESULTS: Marked radiosensitization was obtained with CldC and the three modulators. The average days in tumor regrowth delay for X-ray compared to drugs plus X-ray, respectively, were: PC-3 prostate, 42-97; H-1579 prostate, 29-115; glioblastoma, 5-51; breast, 50-80; lung, 32-123. Comparative studies with PC-3 and H-1579 using CldC coadministered with H4U, showed that both PALA and FdC are dispensable, and the protocol can be simplified with equal and possibly heightened efficacy. For example, PC-3 with X-ray and (1) no drugs, (2) CldC plus the three modulators, (3) a high dose of CldC, and (4) escalating doses of CldC resulted in 0/10, 3/9, 5/10, and 6/9 cures, respectively. The tumor regrowth delay data followed a similar pattern. After treating mice only 11/2 weeks with CldC + H4U, 92% of the PC-3 tumor cells were found to possess CldU in their DNA. The great majority of head-and-neck tumors from patient material had markedly higher levels of dC kinase and dCMP deaminase than found in adjacent normal tissue. Physiologic and histologic studies showed that CldC + H4U combined with X-ray, focused on the cervical spinal cord, did not result in damage to that tissue. CONCLUSIONS: 5-CldC coadministered with only H4U is an effective radiosensitizer of human tumors. Ninety-two percent of PC-3 tumor cells have been shown to take up ClUra derived from CldC in their DNA after only 11/2 weeks and 2 weeks of bolus i.p. injections. Enzymatic alterations that make tumors successful have been exploited for a therapeutic advantage. The great electronegativity, coupled with the relatively small Van der Waal radius of the Cl atom, may result in CldC's possessing the dual advantageous properties of FdC on one hand and BrdU and IdU on the other hand. These advantages include autoenhancing the incorporation of CldUTP into DNA by not only overrunning but also inhibiting the formation of competing TTP pools in tumors. A clinical trial is about to begin, with head-and-neck tumors as a first target of CldC radiosensitization.     

Sensitization to X ray by 5-chloro-2'-deoxycytidine co-administered with tetrahydrouridine in several mammalian cell lines and studies of 2'-chloro derivatives

5-Chloro-2'-deoxycytidine (CldC) + tetrahydrouridine (H4U) sensitizes mammalian cells (HEp-2, RIF-1, S-180) to X ray. This sensitization, as demonstrated previously with HEp-2 cells, is heightened when cells are pre-incubated with inhibitors of pyrimidine synthesis. CHO cells, which intrinsically lack both cytidine deaminase (CD) and deoxycytidylate deaminase (dCMPD), are sensitized to X ray by 5-chlorodeoxyuridine (CldU) but display no significant sensitization with CldC + H4U. The presence and level of these deaminases appears to correlate with X ray sensitization in cell culture. From experiments in cell culture, it can be inferred that one pathway of conversion, deoxycytidine kinase----dCMPD, or CD----thymidine kinase, may be sufficient for metabolizing CldC to a radiosensitizer. However, if both pathways are blocked, as in CHO cells, no X ray sensitization results. In addition to HEp-2 cells, which are extremely elevated in both CD and dCMPD activities, we have examined the sensitization of S-180 and RIF-1 cells to X ray by CldC + H4U. Both cell lines possess an enzymatic profile consistent with their sensitization to X ray by CldC + H4U. Dose enhancement ratios of 1.5 to 1.9 for cells treated with CldC + H4U and ratios of 2.0-2.7 for cells pre-treated with inhibitors of pyrimidine synthesis prior to CldC + H4U have been obtained. Based on reports of the marked X ray sensitization of bacteria by 2'-chloro-2'-deoxythymidine, we obtained 2',5-dichloro-2'-deoxycytidine and 5-bromo-2'-chloro-2-deoxyuridine and found these analogs to be X ray sensitizers of mammalian cells. The strategy that we propose with CldC + H4U and the related 2'-chloro derivatives, based on the elevation of CD and dCMPD in human tumors, offers a degree of selectivity that is not necessarily related to differences in cell kinetics; such that malignancies other than brain tumors may be amenable to this therapy.     

Marked radiosensitization of cells in culture to X ray by 5-chlorodeoxycytidine coadministered with tetrahydrouridine, and inhibitors of pyrimidine biosynthesis

Our approach to overcome the problem of rapid catabolism and general toxicity encountered with 5-halogenated analogues of deoxyuridine (5-bromo, chloro or iododeoxyuridine), which has limited their use as tumor radiosensitizers, is to utilize 5-chlorodeoxycytidine (CldC) with tetrahydrouridine (H4U). We propose that CldC, coadministered with H4U, is metabolized in the following manner: CldC----CldCMP----CldUMP---- ----CldUTP----DNA. All the enzymes of this pathway are elevated in many human malignant tumors and in HEp-2 cells. In X irradiation studies with HEp-2 cells, limited to 1 or 2 radiation doses, we have obtained 3.0 to 3.8 apparent dose enhancement ratios (these represent upper limits) when cells are preincubated with inhibitors of pyrimidine biosynthesis: N-(Phosphonacetyl)-L-aspartate (PALA) and 5-fluorodeoxyuridine (FdU) or 5-fluorodeoxycytidine (FdC) + H4U. Optimum conditions for radiosensitization are: PALA (0.1 mg/ml) 18-20 hr prior to FdU (0.1 microM) or FdC (0.02 microM) + H4U (0.1 mM) followed 6 hr later by CldC (0.1-0.2 mM) + H4U (0.1 mM) for 56-68 hr. Viabilities of 10 +/- 4% to 15 +/- 1% (+/- S.E.) were obtained for drug-treated unirradiated cells. Enzymatic studies indicate that this toxicity may be tumor selective. CldC + H4U alone (at these concentrations) results in 20% substitution of CldU for thymidine in DNA (determined by HPLC analysis). Preliminary toxicity studies indicate that mice will tolerate treatment protocols involving a single dose of PALA (200 mg/kg) followed by a dose of FdU (50 mg/kg) and 3 cycles of CldC (500 mg/kg) + H4U (100 mg/kg) at 10 hour intervals, with marginal weight loss (4%). In this approach we seek to obtain preferential conversion of CldC to CldUTP at the tumor site by taking advantage of quantitative differences in enzyme levels between tumors and normal tissues.     

In vitro and in vivo radiation sensitization by the halogenated pyrimidine 5-chloro-2'-deoxycytidine

5-Chloro-2'-deoxycytidine (Cld/Cyd) is hypothesized to have preferential incorporation into tumor DNA on the basis of elevated deoxycytidine-5'-phosphate deaminase and deoxycytidine kinase levels in tumors. Radiosensitization by Cld/Cyd was evaluated in exponentially growing Chinese hamster ovary cells by determining the ratio of radiation doses in control and treated cells to produce the same degree of cell killing (sensitizer enhancement ratio). Sensitizer enhancement ratios of 1.2-1.8 are seen at Cld/Cyd concentrations of 3-100 microM, 64 h incubation, and 200-600 cGy irradiation. Coincubation with tetrahydrouridine (H4Urd), a proposed inhibitor of Cld/Cyd catabolism by plasma cytidine deaminase resulted in no enhanced drug or radiation cytotoxicity. C3H mice given implants of RIF-1 tumors received 72-h continuous i.p. infusions of Cld/Cyd with or without H4Urd, or 5-bromo-2'-deoxyuridine (BrdUrd). Excised tumors were irradiated as single cell suspensions in vitro. Infusions of equimolar (0.4 mmol/kg/day) Cld/Cyd or BrdUrd resulted in greater radiosensitization by BrdUrd with no potentiation of Cld/Cyd by coinfusion with 0.8 mmol/kg/day H4Urd. Infusions with equitoxic doses of Cld/Cyd (0.8 mmol/kg/day) or BrdUrd (0.4 mmol/kg/day) yielded equal BrdUrd and Cld/Cyd sensitizer enhancement ratios of 1.6, without H4Urd potentiation of Cld/Cyd. Fluorescence-activated cell sorter analysis of tumor cell suspensions using a monoclonal antibody reactive with BrdUrd and Cld/Cyd disclosed a population of noncycling cells in tumors treated with Cld/Cyd/H4Urd that is not seen in tumors exposed to either BrdUrd or Cld/Cyd alone.     

Depression of DNA synthesis in mouse spleen after treatment with 5-aza-2'-deoxycytidine.

5-Aza-2'-deoxycytidine is a highly effective cytostatic agent that preferentially affects the lymphatic system. Pretreatment of noninbred H mice with the drug markedly depressed the level of thymidine (dThd) incorporation into DNA in the spleen and also lowered the dThd and thymidylate kinase activities. Maximum effects were observed following administration of the analog in a single dose 24 hours before the mice were killed. Whereas cytidine and dThd did not reverse the inhibitory effect of 5-aza-2'-deoxycytidine, excessive doses of deoxycytidine partially reversed this inhibition. Similar to the depression of dThd incorporation, a depression in the incorporation of deoxycytidine and cytidine into spleen DNA was found after 24-hour pretreatment with 5-aza-2'-deoxycytidine. However, 7 days following 5-aza-2'-deoxycytidine treatment, the incorporation of dThd into DNA in the spleens of mice was significantly increased. [3H]5-aza-2'-deoxycytidine was rapidly incorporated into spleen DNA, whereas deoxycytidine interfered with the incorporation of [3H]5-aza-2'-deoxycytidine.     

Protective, tumor-selective dual pathway activation of 5-fluoro-2'-deoxycytidine provided by tetrahydrouridine in mice bearing mammary adenocarcinoma-755

Treatment of C57BL X DBA/2 F (hereafter called BD2F) mice bearing ascitic mammary adenocarcinoma-755 (ADC-755) with [3H]-5-fluoro-2'-deoxycytidine ([3H]FdCyd) plus tetrahydrouridine (H4Urd) resulted in antimetabolite pool sizes indicative of a tumor-selective, dual pathway metabolism of FdCyd via both cytidine deaminase and deoxycytidine kinase. In contrast to the high levels of all RNA- and DNA-level antimetabolites (as assayed by high performance liquid chromatography) derived from FdCyd found in tumor tissue, normal tissues (bone marrow, intestine, liver, and spleen) and serum metabolized FdCyd to only a small extent following FdCyd plus H4Urd treatment. RNA-level antimetabolite pools and 5-fluoro-2'-deoxyuridine (FdUrd) were generally 100-fold lower in normal than in tumor tissue, and 5-fluoro-2'-deoxyuridylate was 10- to 15-fold lower in normal than in tumor tissue. The use of [3H]FdUrd, on the other hand, resulted in the formation of higher levels (10- to 40-fold) of DNA- and RNA-level antimetabolites in normal tissue and lower levels (1/8) of 5-fluoro-2'-deoxyuridylate in tumor tissue. Both [3H]FdCyd plus H4Urd and [3H]FdUrd were utilized at their optimal drug doses. FdUrd- and FdCyd-derived metabolic products incorporated into the RNA and DNA of normal and tumor tissue of BD2F mice bearing ADC-755 were also examined. The drug combination [3H]FdCyd plus H4Urd resulted in the selective incorporation of antimetabolites into tumor RNA and DNA; only a very small extent of antimetabolites incorporated into normal tissue RNA and DNA. FdCyd was incorporated 5- to 10-fold greater in tumor than intestine, liver, or spleen following FdCyd plus H4Urd administration. FdCyd incorporation was 190-fold greater in tumor than in bone marrow. Mice bearing ADC-755 treated with [3H]-FdUrd resulted in only marginal selectivity in terms of antimetabolite incorporation in tumor tissue. Deoxycytidylate and cytidine deaminase enzyme assays have confirmed that H4Urd administration effectively inhibited normal cytidine deaminase activities, while only weakly inhibiting the elevated levels found in tumor tissue. Thymidine kinase, deoxycytidine kinase, deoxycytidylate deaminase, and cytidine deaminase have been shown previously to be significantly elevated in the mouse tumor model used; these enzymatic elevations are also characteristic of many human tumors. Treatment with FdCyd plus H4Urd resulted in 17 of 30 cures against ADC-755 compared to 4 of 20 and 0 of 20 for 5-fluorouracil and 5-fluoro-2'-deoxyuridine treatments, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Low cytidine deaminase levels in human hematopoietic cell lines

Purine and pyrimidine enzyme profiles of human cell lines have been investigated. A novel observation was the finding that most of the cell lines showed very low or undetectable levels of cytidine (deoxycytidine) deaminase, while they possessed pyrimidine 5'-nucleotidase, cytidine and deoxycytidine kinase activities. Most cell lines showed high levels of adenosine deaminase and purine nucleoside phosphorylase activities and low levels of purine 5'-nucleotidase. We propose that high adenosine deaminase and purine nucleoside phosphorylase activities and low cytidine deaminase activity may be of importance for immature hematopoietic cells in order to ensure a balanced synthesis of the DNA precursors.     

Cytotoxic and biochemical effects of thymidine and 3-deazauridine on human tumor cells

Cytotoxicity and perturbations of the deoxyribonucleoside triphosphate pools caused by thymidine were studied in thymidine-sensitive and -resistant human tumor cells. Incubation with 1 mM thymidine reduced cell viability by more than 90% in the three sensitive cell lines (two melanomas and one adrenal carcinoma) and reduced the growth rate without decreasing the viability of resistant LO melanoma cells. Thymidine (1 mM) greatly increased the ratio of the deoxythymidine 5'-triphosphate to deoxycytidine 5'-triphosphate pools in the sensitive cells compared to LO cells and also caused larger relative increases in the pool sizes of deoxyguanosine 5'-triphosphate and deoxyadenosine 5'-triphosphate in the sensitive compared to the resistant cells. 3-Deazauridine, known to inhibit synthesis of deoxycytidine 5'-triphosphate and cytidine 5'-triphosphate in other cell lines, potentiated the cytotoxicity of thymidine for thymidine-sensitive BE melanoma and LO cells. In LO cells, 3-deazauridine (50 microM) decreased the intracellular pool of deoxycytidine 5'-triphosphate to the level obtained with 1 mM thymidine. Lower concentrations of deoxycytidine as compared to cytidine were required to protect BE and LO cells against the cytotoxicity of thymidine plus 3-deazauridine. Deoxycytidine also was more effective than was cytidine in preventing loss of cell viability after exposure to thymidine or to 3-deazauridine individually. In these human melanoma cells, ribonucleotide reductase may be a major site of action of thymidine, of 3-deazauridine, and of both drugs in combination. These results indicate that in human tumor cells the cytotoxic effect of thymidine correlates with greater perturbations of the pyrimidine deoxyribonucleoside 5'-triphosphate pools and that thymidine and 3-deazauridine, which independently reduce the intracellular levels of deoxycytidine 5'-triphosphate, act synergistically against human tumor cells.     

Biochemical changes associated with the development of resistance to 5-azacytidine in AKR leukemic mice

Resistance to 5-azacytidine, a potent inhibitor of the growth of mouse lympaoid leukemia cells in vivo, was achieved in an inbred strain of AKR mice in the third transplant period. The incorporation of orotic acid into the livers of such resistant mice was more than six times lower than in animals carrying the 5-azacytidine-sensitive parent strain of leukemia. The incorporation of orotic acid by 5-azacytidine-sensitive mice was inhibited by 5-azacytidine twice as much as in 5-azacytidine-resistant animals. The incorporation of uridine, cytidine, thymidine, deoxycytidine, and of guanosine into nucleic acids was also significantly depressed in 5-azacytidine-resistant leukemic cells. Uridine kinase activity was decreased by 20%, while uridine phosphorylase activity was diminished by 31–50%. The resistant leukemic mice were cross-resistant to 5-fluorouracil; in contrast, however, their sensitivity to aminopterin was increased significantly.     

Pyrimidine nucleotide synthesis is more extensive in poorly differentiated than in well-differentiated human gastric carcinoma

In tissues obtained from patients undergoing gastrectomy, the activities of 12 enzymes involved in pyrimidine nucleotide synthesis: cytidine triphosphate (CTP) synthetase, deoxycytidine monophosphate (dCMP) deaminase, thymidine monophosphate (dTMP) kinase, uridine (Urd), deoxycytidine (dCyd) and thymidine (dThd) kinases, Urd, deoxyuridine (dUrd) and dThd phosphorylases, cytidine (Cyd) and dCyd deaminases, and DNA polymerase were examined in the eight-well-differentiated and 12 poorly differentiated gastric cancer tissues and the ten normal tissues. These cases were clinically advanced and serosal invasions were evident. Activities of these enzymes were higher in the poorly differentiated tissues than the well differentiated type and in the normal tissues. Significant differences were noted between the poorly differentiated and well-differentiated types, in dTMP kinase (P less than 0.02), dThd kinase (P less than 0.05), dThd phosphorylase (P less than 0.01), and DNA polymerase (P less than 0.05). The authors' findings show that the level of pyrimidine nucleotide synthesis, in both de novo and salvage pathways, is higher in the poorly differentiated gastric cancer tissues than in the well-differentiated type and suggest that antitumor drugs have an increased susceptibility in cases of poorly differentiated gastric carcinoma.     

Tumor radiosensitization by sustained intratumoral release of bromodeoxyuridine.

We have previously reported that the use of the polymer bis(p-carboxyphenoxy)propane-sebacic acid (20:80) for intratumoral delivery of cis-platinum in a mouse tumor model (RIF-1) potentiated the effects of acute and fractionated radiation. This mode of drug delivery seems particularly applicable to the administration of radiosensitizing drugs because an optimum concentration of radiosensitizer can be maintained in the tumor over the prolonged period required for fractionated radiation treatment. We have now investigated, in the same tumor model, radiosensitization by the thymidine analogue bromodeoxyuridine (BrdUrd). BrdUrd (20%, w/w) was incorporated into bis(p-carboxyphenoxy)propane-sebacic acid (20:80) and polymer rods containing the drug implanted in the RIF-1 tumor. Preliminary in vitro studies of the rate of release of BrdUrd from the polymer showed an initial rapid loss over 24 h, followed by a slower release extending over the next 5 days. In experiments in which tumor cells, which had incorporated BrdUrd in vivo from implanted polymer, were excised and a single cell suspension irradiated in vitro radiosensitization indicative of BrdUrd incorporation was associated mainly with an increase in the alpha constant for the linear quadratic model of cell survival. Radiosensitization was seen for tumor cells harvested between 5 and 10 days after polymer implant, a finding that is consistent with results of experiments in which the percentage of cells that had incorporated BrdUrd were measured by flow cytometry at various times after polymer/BrdUrd implant. The proportion of tumor cells positive for BrdUrd was 40-50% between 3 and 8 days after polymer implant. When tumors were irradiated in situ and response measured in terms of tumor growth delay (TGD), radiosensitization was not seen for an acute dose of 16.5 Gy. In contrast, significant radiosensitization was seen for fractionated treatments when polymer/BrdUrd was implanted 3 days before the first radiation dose. For a dose of 5 x 6 Gy, TGD was increased from 22 days for radiation alone to 27 days for radiation plus polymer implant. For 10 x 6 Gy fractions, TGD increased from 45-77 days for those mice in whom the tumor eventually regrew, whereas for 25% of the mice in this group the tumor volume was reduced to a point where it was no longer detectable and there was no recurrence for at least 120 days after treatment.     

Analysis of the drug synergism between thymidine and arabinosyl cytosine using mouse S49 T lymphoma mutants

Summary The synergism between arabinosyl cytosine (araC) and thymidine is characterized using two mutant S49 T lymphoma cell populations with altered deoxyribonucleotide metabolism. AraC-1-6 cells are deficient in dCMP deaminase activity resulting in a secondary elevation of intracellular dCTP pools, whereas dGuo-200-l cells have a mutation in the M₁ subunit of ribonucleotide diphosphate reductase, which also results in elevation of dCTP levels. These two mutant cell populations are partially resistant to araC cytotoxicity as compared to the wild type cells. The resistance to araC is contributed to the elevation of dCTP levels in these mutants which prevent araC incorporation into the DNA due to feedback inhibition of deoxycytidine kinase. Addition of extracellular thymidine to dCMP deaminase deficient cells causes a decrease in dCTP levels and in parallel increase their sensitivity to araC. In contrast, extracellular thymidine does not reduce dCTP levels in the mutant cells with altered ribonucleotide reductase and no synergism between araC and thymidine is observed in these cells. The expansion of dTTP pools in the presence of thymidine is similar in the two mutants. These results suggest that the depletion of dCTP pools by thymidine is responsible for the synergistic action of thymidine on araC cytotoxicity and that dTTP does not directly enhance the incorporation of araC into the DNA of T lymphoma cells.     

Comparison of pyrimidine nucleotide synthetic enzymes involved in 5-fluorouracil metabolism between human adenocarcinomas and squamous cell carcinomas

The activities of orotate phosphoribosyltransferase (OPRT), cytidine triphosphate (CTP) synthetase, deoxycytidine monophosphate (dCMP) deaminase, thymidine monophosphate (dTMP) kinase, uridine (Urd) kinase, thymidine (dThd) kinase, Urd and dThd phosphorylases, and DNA polymerase were examined in the eight human lung squamous cell carcinomas and five lung adenocarcinomas, and five tumor-adjacent normal lung tissues. All of these enzymes are involved in pyrimidine nucleotide synthesis. The metabolism of 5-fluorouracil (5-FU) was determined. The levels of these enzymes, except for OPRT, were high in tumor tissues and almost the same between lung squamous cell carcinomas and adenocarcinomas, with no statistical difference. The activities for phosphorylation and degradation of 5-FU were similar in each tissue type of tumor. As 5-FU is incorporated into tumor cells and is metabolized actively to 5-FU nucleotides in squamous cell carcinoma tissues, at almost the same level seen in adenocarcinoma tissues, this drug should have a wide clinical application.     

Fluorodeoxyuridine-mediated modulation of iododeoxyuridine incorporation and radiosensitization in human colon cancer cells in vitro and in vivo.

A study was conducted to assess the potential of 5-fluoro-2'-deoxyuridine (FdUrd) to increase the incorporation and radiosensitizing properties of 5-iodo-2'-deoxyuridine (IdUrd) using HT29 human colon cancer cells both in vitro and in nude mice bearing these tumors as xenografts. The purpose of this study was to assess (a) whether FdUrd could increase IdUrd efficacy using clinically achievable concentrations of drugs; (b) the relationships among radiosensitization, DNA damage and repair, and analogue incorporation; and (c) whether FdUrd improved the selectivity of IdUrd incorporation into tumor cells compared to normal tissues. It was found that FdUrd, at clinically achievable concentrations (1-100 nM), significantly increased IdUrd incorporation under all conditions but particularly when the IdUrd concentration was less than or equal to 10 microM. FdUrd increased IdUrd-mediated radiosensitization in proportion to the increase in IdUrd incorporation. FdUrd potentiated the ability of IdUrd to increase radiation-induced DNA double-strand breaks and to slow their repair. When IdUrd alone (100 and 200 mg/kg/day) was infused into nude mice bearing tumors, the extent of thymidine replaced in the tumor was 1.6 +/- 0.4 (mean +/- SE) and 2.5 +/- 0.4%, respectively. The combination of FdUrd (0.1 mg/kg/day) and IdUrd (100 mg/kg/day) increased the incorporation in the tumor to 5.3 +/- 0.9% with less toxicity than resulted from the use of 200 mg/kg/day of IdUrd alone. These data show that FdUrd is an effective biomodulator, because, for the same extent of normal tissue incorporation, the combination of IdUrd and FdUrd produces significantly greater incorporation into the tumor compared to the use of IdUrd alone. Furthermore, they suggest that the regional application of FdUrd with IdUrd, either through the use of regional infusions or in combination with focused irradiation, could potentially improve the outcome of treatment of localized gastrointestinal cancer.     

DNA synthesis and DNA polymerase activity in Leydig cells of diethylstilbestrol-stimulated mouse testes

Using a modification of the collagenase dispersion method of Dufau et al., we examined changes in DNA synthesis produced by estrogens in the interstitial cells of mice that develop malignant Leydig cell tumors after prolonged estrogen administration. Previous work in cryptorchid mice indicated that during continuous estrogen administration [3H]thymidine incorporation into DNA rises to a maximum in 3 to 4 days and then falls to approximately base levels within 2 to 3 weeks. This was confirmed both in Leydig cell concentrates of estrogen-treated mice after either injection with [3H]thymidine or incubation with [3H]thymidine in vitro. This DNA synthesis was blocked by hydroxyurea. DNA synthesis in cells of estrogen-treated BALB/c mice of the Huseby substrain, which have a high incidence of Leydig cell tumors, was 5 to 11 times that in untreated controls. Cells from estrogen-treated C3H/Bi mice, which have a low incidence of Leydig cell tumors, showed only a 2- to 3-fold increase. In the Huseby substrain the rise of DNA synthesis is a peak and subsequent recession were paralleled by a rise and fall in DNA polymerase alpha activity. DNA polymerase beta did not show this variation. In C3H/Bi mice, neither polymerase showed significant change. The evidence suggests that the early estrogen-stimulated DNA synthesis is probably replicative and is associated with increased DNA polymerase alpha activity.     

Modulation of iododeoxyuridine-mediated radiosensitization by 5-fluorouracil in human colon cancer cells.

The incorporation of 5-iodo-2'-deoxyuridine (IdUrd), a thymidine analog radiosensitizer, can be increased by the use of modulators such as 5-fluorouracil (FUra). FUra is a particularly attractive potential modulator to use against colorectal cancer, as it is the most active single agent in the treatment of this disease. To begin to define the conditions for the optimal combination of IdUrd and FUra in the treatment of patients with colorectal cancer, a study was conducted of the effect of FUra on IdUrd-mediated radiosensitization in cultured HT29 human colon cancer cells. It was found that when cells were exposed to concentrations of IdUrd typical of those obtained through intravenous exposure (1-3 microM), FUra (1 microM) increased radiosensitization beyond that which would be predicted for the same extent of incorporation produced by incubation with IdUrd alone. This increase appeared to result from a combination of at least two effects: FUra-mediated cell cycle redistribution and increased IdUrd incorporation. When a higher concentration of IdUrd (10 microM) was used with FUra (1 microM), cell cycle distribution returned to nearly normal, and radiosensitization was equal to that predicted by the extent of incorporation of IdUrd. These data demonstrate that the combination of FUra and IdUrd can produce radiosensitization both through increased IdUrd incorporation and cell cycle redistribution. Furthermore, they suggest that, in the presence of a modulator, it may not be necessary to achieve high levels of IdUrd incorporation to produce significant tumor radiosensitization.     

Distribution of 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl) uracil in mice bearing colorectal cancer xenografts: rationale for therapeutic use and as a positron emission tomography probe for thymidylate synthase.

PURPOSE: In colorectal, breast, and head and neck cancers, response to 5-fluorouracil is associated with low expression of thymidylate synthase. In contrast, tumors with high expression of thymidylate synthase may be more sensitive to prodrugs such as 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl) uracil (FAU) that are activated by thymidylate synthase. These studies were designed to evaluate FAU as a potential therapeutic and diagnostic probe. EXPERIMENTAL DESIGN: [18F]-FAU and [3H]-FAU were synthesized with >97% radiochemical purity. [3H]-FAU or [18F]-FAU was administered intravenously to severe combined immunodeficient mice bearing either HT29 (low thymidylate synthase) or LS174T (high thymidylate synthase) human colon cancer xenografts. Four hours after [3H]-FAU dosing, tissue distribution of total radioactivity and incorporation of 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl) 5-methyluracil (FMAU), derived from thymidylate synthase activation of FAU, into tumor DNA was measured. Positron emission tomography (PET) images were obtained for 90 minutes after injection of [18F]-FAU. Thymidylate synthase activity was determined in vitro in tumors from untreated mice by [3H] release from [3H]dUMP. Each cell line was incubated in vitro with [3H]-FAU or [3H]-FMAU in the absence or presence of 5-fluoro-2'-deoxyuridine (FdUrd) and then was analyzed for incorporation of radiolabel into DNA. RESULTS: Thymidylate synthase enzymatic activity in LS174T xenografts was approximately 3.5-fold higher than in HT29 xenografts, and incorporation of radioactivity derived from [3H]-FAU into LS174T DNA was approximately 2-fold higher than into HT29 DNA. At 240 minutes, radioactivity derived from [3H]-FAU was approximately 2-fold higher in tumors than in skeletal muscle. At times up to 90 minutes, PET imaging detected only small differences in uptake of [18F]-FAU between the tumor types. Fluorine-18 in skeletal muscle was higher than in tumor for the first 90 minutes and plateaued earlier, whereas [18F] in tumor continued to increase during the 90-minute imaging period. For both cell lines in vitro, FdUrd decreased the rate of incorporation of [3H]-FAU into DNA, whereas the incorporation of [3H]-FMAU was increased. CONCLUSIONS: These results for FAU incorporation into DNA in vitro and in vivo further support clinical evaluation of FAU as a therapeutic agent in tumors with high concentrations of thymidylate synthase that are less likely to respond to 5-fluorouracil treatment. The high circulating concentrations of thymidine reported in mice may limit their utility in evaluating FAU as a PET probe.     

Modulation of the effect of 1-beta-D-arabinofuranosylcytosine based on changes of cytidine deaminase activity in HL60 cells

The effect of changes in cytidine deaminase activity on the growth inhibitory effect of 1-beta-D-arabinofuranosylcytosine (Ara-C) has been investigated in the human promyelocytic cell line, HL60. 1,25 Dihydroxy vitamin D3 (vit. D3), which is an inducer of differentiation, caused an increase of cytidine deaminase in HL60 cells simultaneous with an inhibition of the effect of Ara-C. On the other hand, retinoic acid, which also induces differentiation had no influence on either the activity of cytidine deaminase or the effect of Ara-C. An atoxic inhibitor of cytidine deaminase tetrahydrouridine, THU, enhanced the effect of Ara-C. THU also enhanced the effect of Ara-C, even if the Ara-C effect was inhibited by deoxycytidine. These results show that it is possible to modulate the effect of Ara-C by changing the activity of cytidine deaminase. Furthermore, changes in the activity of cytidine deaminase and the effect on the Ara-C growth inhibition vary dependent on the differentiating inducer used.     

Effect of cyclophosphamide on the pathophysiology of RIF-1 solid tumors

The present experiments were conducted to determine the effects of cyclophosphamide (150 mg/kg) on the pathophysiology of RIF-1 solid tumors and to determine the temporal relationship between treatment mediated changes in tumor vascular physiology, cell proliferation, and chemoresponsiveness in vivo. Capillary permeability and plasma and extracellular water volumes were determined by a 125I-bovine serum albumin, 51Cr-EDTA double isotope dilution assay at various intervals after cyclophosphamide. Tumor blood flow and exchangeable erythrocyte vascular volumes were determined by 86RbCl distribution and 51Cr-labeled erythrocyte dilution methods. Cell proliferation in RIF-1 tumors, assessed by [3H]thymidine labeling index and tumor growth fraction (primer-dependent DNA polymerase labeling assay) measurements, was inhibited for up to 3 days by cyclophosphamide. Although tumor regrowth was not apparent until Day 10, cell kinetic studies indicated proliferative recovery in the surviving cell population on Days 4 and 5 after treatment. Increases in tumor blood flow and tumor vascular volumes were temporally coincident with this proliferative response. In split-dose experiments, the time-dependent increases in the chemoresponsiveness of RIF-1 tumors, after cyclophosphamide, may be due not only to the increased proliferation of repopulating cells, but also to vascular responses attendant with cytoreduction.     

Selective radiosensitization and cytotoxicity of human melanoma cells using halogenated deoxycytidines and tetrahydrouridine

The halogenated pyrimidines 5-chloro-2'-deoxycytidine (CldCyd) and 5-bromo-2'-deoxycytidine (BrdCyd) can act as radiosensitizers and cytotoxic agents. It was hypothesized that tumor cells and normal cells might use different metabolic pathways to incorporate these halogenated deoxycytidines into DNA. This difference could potentially be exploited to produce selective radiosensitization and cytotoxicity of human tumor cells compared to normal human fibroblasts. This hypothesis was tested using two human melanoma cell lines and two normal fibroblast cell lines. Either CldCyd or BrdCyd alone caused both cytotoxicity and radiosensitization of tumor and normal cells. The addition of the cytidine deaminase inhibitor tetrahydrouridine (H4U) significantly protected the normal cells but had relatively little effect on the tumor cells. These data indicate that it may be possible to exploit differences between the pyrimidine metabolism of normal cells and melanoma cells to improve the therapeutic index of halogenated pyrimidines both as radiosensitizers and as cytotoxic agents.