Sheldon Greer

DNA mismatch repair (MMR)-dependent 5-fluorouracil cytotoxicity and the potential for new therapeutic targets

The metabolism and efficacy of 5‐fluorouracil (FUra) and other fluorinated pyrimidine (FP) derivatives have been intensively investigated for over fifty years. FUra and its antimetabolites can be incorporated at RNA‐ and DNA‐levels, with RNA level incorporation provoking toxic responses in human normal tissue, and DNA‐level antimetabolite formation and incorporation believed primarily responsible for tumour‐selective responses. Attempts to direct FUra into DNA‐level antimetabolites, based on mechanism‐of‐action studies, have led to gradual improvements in tumour therapy. These include the use of leukovorin to stabilize the inhibitory thymidylate synthase‐5‐fluoro‐2′‐deoxyuridine 5′ monophoshate (FdUMP)‐5,10‐methylene tetrahydrofolate (5,10‐CH2FH4) trimeric complex. FUra incorporated into DNA also contributes to antitumour activity in preclinical and clinical studies. This review examines our current state of knowledge regarding the mechanistic aspects of FUra:Gua lesion detection by DNA mismatch repair (MMR) machinery that ultimately results in lethality. MMR‐dependent direct cell death signalling or futile cycle responses will be discussed. As 10–30% of sporadic colon and endometrial tumours display MMR defects as a result of human MutL homologue‐1 (hMLH1) promoter hypermethylation, we discuss the use and manipulation of the hypomethylating agent, 5‐fluorodeoxycytidine (FdCyd), and our ability to manipulate its metabolism using the cytidine or deoxycytidylate (dCMP) deaminase inhibitors, tetrahydrouridine or deoxytetrahydrouridine, respectively, as a method for re‐expression of hMLH1 and re‐sensitization of tumours to FP therapy.

Incorporation of 5-Substituted Analogs of Deoxycytidine into DNA of Herpes Simplex Virus-Infected or -Transformed Cells Without Deamination to the Thymidine Analog

The incorporation into DNA of 5-bromocytosine and 5-iodocytosine, derived from their respective administered deoxyribonucleoside analogs, has been demonstrated in studies with cells infected with herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) and in cells transformed with the thymidine kinase gene of HSV-1. No significant incorporation of iodocytosine or iodouracil occurred in the DNA of uninfected or nontransformed cells when the deaminating enzymes were inhibited, in accord with past studies in our laboratory with 5-bromodeoxycytidine and tetrahydrouridine. When 2′-deoxytetrahydrouridine, a potent inhibitor of cytidine deaminase and dCMP deaminase, was utilized, all the counts in DNA that were derived from [125I]iododeoxycytidine appeared as iodocytosine in HSV-infected cells. In the absence of a deaminase inhibitor, 32 to 45% of the counts associated with DNA pyrimidines appeared as iodocytosine, and 55 to 68% appeared as iodouracil in HSV-infected cells. Substantial incorporation of iodocytosine (16%) occurred in cells transformed with the HSV thymidine kinase gene, suggesting the importance of the specificity of cellular nucleoside kinases and the activity of the deaminases in presenting unmodified bases to an undiscriminating polymerase. Incorporation into DNA of bromocytosine derived from [3H]bromodeoxycytidine was demonstrated in HSV-2 infected cells; very little incorporation of bromocytosine compared with bromouracil could be demonstrated in these cells in the absence of inhibition of the deaminases (19% of the total counts associated with pyrimidines with deaminase inhibition and 1.5% without). Limited studies with 5-methyl[5-3H]deoxycytidine indicated essentially no (or very little) incorporation of this analog as such in the DNA of HSV-1- and HSV-2-infected and -transformed cells. This suggests an exclusion or repair mechanism preventing inappropriate methylcytosine incorporation in DNA. The addition of nucleoside and deoxyribonucleoside deaminase inhibitors, which leads to the incorporation of 5-halogenated analogs of deoxycytidine into DNA as such, does not impair their antiviral activity. We infer from studies with 4-N-alkyl (ethyl and isopropyl)-substituted analogs of iododeoxycytidine that they are incorporated as such into DNA without deamination and effectively inhibit the virus at concentrations that are marginally toxic. Among the several reasons presented for the heightened potential efficacy of analogs of deoxycytidine compared with those of deoxyuridine is that the former, as analogs of 5-methyldeoxycytidine, may impair viral replication by perturbing processes involving methylation and changes in the methylation of deoxycytidine in DNA which appear to be important for the process of HSV maturation. In addition, this capacity to perturb methylation may, in turn, be the key to their potential as agents affecting entry into or emergence from latency, a process in which dramatic changes in the postpolymer 5-methylation of deoxycytidine occur in the DNA of herpesviruses.

Host-cell reactivation of UV-irradiated and chemically-treated herpes simplex virus-1 by xeroderma pigmentosum, XP heterozygotes and normal skin fibroblasts.

Abstract The host-cell reactivation of UV-irradiated and N -acetoxy-2-acetylamino-fluorene-treated herpes simplex virus type 1 strain MP was studied in normal and xeroderma pigmentosum human skin fibroblasts. Virus treated with either agent demonstrated lower survival in XP cells from complementation groups A, B, C and D than in normal fibroblasts. The relative reactivation ability of XP cells from the different genetic complementation groups was found to be the same for both irradiated and chemically treated virus. In addition, the inactivation kinetics for virus treated with either agent in the XP variant were comparable to that seen in normal skin fibroblasts. The addition of 2 or 4 mmoles caffeine to the post-infection assay medium had no effect on the inactivation kinetics of virus treated by either agent in the XP variant or in XP cells from the different genetic complementation groups. Treatment of the virus with nitrogen mustard resulted in equivalent survival in normal and XP genetic complementation group D cells. No apparent defect was observed in the ability of XP heterozygous skin fibroblasts to repair virus damaged with up to 100 μg N -acetoxy-2-acetylaminofluorene per ml. These findings indicate that the repair of UV-irradiated and N -acetoxy-2-acetylaminofluorene-treated virus is accomplished by the same pathway or different pathways sharing a common intermediate step and that the excision defect of XP cells plays little if any role in the reactivation of nitrogen mustard treated virus.

Viruses of Cariogenic Streptococci

Eight cariogenic strains of Streptococcus mutans and one cariogenic strain of Streptococcus salivarius lysed after exposure to ultraviolet light, mitomycin, nitrosoguanidine, and streptomycin. Each of these strains released a virus with a similar morphology after ultraviolet light and mitomycin induction. Neither lysis nor a virus was demonstrated in several noncariogenic strains of streptococci.

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.

Radiation, pool size and incorporation studies in mice with 5-chloro-2′-deoxycytidine

Bolus doses of 5-chlorodeoxycytidine (CldC) administered with modulators of pyrimidine metabolism, followed by X-irradiation, resulted in a 2-fold dose increase effect against RIF-1 tumors in C3H mice. Pool size studies of the fate of [14C]-CldC in BDF1 mice bearing Sarcoma-180 tumors, which demonstrated the rapid formation of 5-chlorodeoxycytidylate (CldCMP), and incorporation of CldC as such in RIF-1 tumor DNA, indicate that CldC is a substrate for deoxycytidine kinase, as our past Km studies have shown. Our data indicate that 5-chlorodeoxyuridine triphosphate (CldUTP) accumulates from both the cytidine deaminase-thymidine kinase pathway, as well as from the deoxycytidine kinase-dCMP deaminase pathway, in tumor tissue. As shown in a previous study, tetrahydrouridine (H4U), a potent inhibitor of cytidine deaminase, can effectively inhibit the enzyme in the normal tissues of BDF1 mice. When H4U was administered with the modulators N-(phosphonacetyl)-L-aspartic acid (PALA) and 5-fluorodeoxycytidine (FdC), the levels of CldC-derived RNA and DNA directed metabolites increased in tumor and decreased in normal tissues compared to when CldC was administered alone. These modulators inhibit the de novo pathway of thymidine biosynthesis, lowering thymidine triphosphate (TTP) levels, which compete with CldUTP for incorporation into DNA. 5-Benzylacyclouridine (BAU), an inhibitor of uridine phosphorylase, was also utilized. DNA incorporation studies using C3H mice bearing RIF-1 tumors showed that the extent of incorporation of 5-chlorodeoxyuridine (CldU) into DNA correlates with the levels of cytidine and dCMP deaminases; this is encouraging in view of their high activity in many human malignancies and the low activities in normal tissues, including those undergoing active replication. Up to 3.9% replacement of thymidine by CldU took place in RIF-1 tumors, whereas incorporation into bone marrow was below our limit of detection. CldC did not result in photosensitization under conditions in cell culture in which radiosensitization to X rays was obtained. Thus, the combination of CldC with modulators of its metabolism has potential as a modality of selective radiosensitization for ultimate clinical use in a wider range of tumors than those of the brain.

An assay for pyrimidine deoxyribonucleoside kinase using γ-32P-labeled ATP

Abstract A rapid, simple and sensitive assay for pyrimidine deoxyribonucleoside kinase is described. After phosphorylation of unlabeled nucleoside substrate through the transfer of the γ-phosphate of [γ- 32 P]ATP, the reaction mixture is subjected to 1 n HCl at 100°C. The β- and γ-phosphates of unreacted ATP are hydrolyzed to inorganic phosphate while the 5′-phosphate of the pyrimidine deoxyribonucleotide product is not hydrolyzed. Radioactive phosphate remaining in the supernatant fluid after precipitation of inorganic phosphate corresponds to product and is measured by liquid scintillation spectrometry. Evidence is presented suggesting that pyrimidine and purine ribonucleoside kinase activity can also be determined by this assay.

Methylation of HSV-1 DNA as a mechanism of viral inhibition: Studies of an analogue of methyldeoxycytidine: Trifluoromethyldeoxycytidine (F3mdCyd)

Although several hypomethylating agents such as 5-azadeoxycytidine and 5-fluorodeoxycytidine have been shown to activate transcription after incorporation into viral or cellular DNA, agents which selectively affect the methylation status of virus-infected cells have not been described. Studies on the antiviral effect of the methyldeoxycytidine (mdCyd) analogue trifluoromethyldeoxycytidine (F3mdCyd) showed significant antiviral activity against herpes simplex virus type 1 (HSV-1). This analogue of both dCyd and dThd is selectively incorporated into the DNA of herpesvirus infected cells due to the unique specificity of the herpesvirus thymidine kinase (TK) because the HSV-1 TK is both a dCyd and dThd kinase. In contrast, the deoxycytidine kinase of uninfected cells preferentially phosphorylates dCyd and has a poor affinity for F3mdCyd. F3mdCyd hemisubstituted M13 DNA displayed the same properties as mdCyd-substituted M13 DNA with respect to cleavage by restriction enzymes, and acted as an efficient template for eukaryotic DNA methyltransferase (S-adenosyl-L-methionine DNA (cytosine-5) methyltransferase: EC 2.1.1.37). Using the persistently infected CEM cell model system, the extent of DNA methylation was shown to increase in a dose-related manner when HSV-1-infected CEM cells were treated with increasing concentrations of F3mdCyd. Higher levels of methylation correlated with significant decreases in HSV-1 titers. Isoschizomer analyses followed by Southern blotting and hybridization with genomic HSV-1 DNA showed that DNA from HSV-1-infected, analogue-treated Vero cells was resistant to cleavage by restriction enzymes at a time when productive virus was not present in culture. We infer from these results that the methylation-like properties of the incorporated F3mdCyd occur concomitantly with, and appear to be involved in, the mechanisms of the analogues antiviral effect towards HSV-1.

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

PURPOSE The studys 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 CldCs 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.

Enzyme-Driven Chemo-and Radiation-Therapy with 12 Pyrimidine Nucleoside Analogs Not Yet in the Clinic

Enzymatic activity from tumor and adjacent normal tissue of 200 patients involving deoxycytidine kinase (dCK), uridine/cytidine kinase (U/CK), cytidine deaminase (CD) and deoxycytidylate deaminase (dCMPD) was quantified. Patients with brain (17), colon (24), and breast (30) tumors, 53, 67, and 73%, respectively, had an elevated T/N value (Specific Activity of tumor/ Specific Activity of normal tissue) involving dCK and dCMPD suggesting chemotherapy with 5-fluorodeoxycytidine (5-FdC) alone or in combination with thymidine plus deoxytetrahydrouridine, or with the radiosensitizer, 5-chlorodeoxycytidine (5-CldC) plus tetrahydrouridine (H4U). Among patients with colon (19) and pancreatic tumors (40), 53 and 68 %, respectively, displayed T/N values >4 for CD suggesting chemotherapy with 5-FdC, 4-N-methylamino-5-FdC, 5-trifluoromethyldeoxycytidine and radiosensitization with 5- CldC, 4-N-methylamino-5-CldC, 5-iododeoxycytidine and 5-bromodeoxycytidine. The percent of patients with tumors with a T/N value >4 for U/CK in lung (72), colon (23) and breast (28) was 47, 61 and 68, respectively, suggesting zebularine (plus thymidine) treatment for tumors involving gene silencing. Evidence is presented that the 4-N-alkylamino-dC substituted nucleosides and those with large 5-substitutions are activated only via CD to thymidine kinase (TK) using end-points of cytotoxicity and/or radiosensitization: H4U, the inhibitor of CD is an antagonist, cells with low CD or no TK are resistant to the analogs, the end points are indifferent to the dCK status of cells, they are poor substrates for dCK and good substrates for CD, whereas 5-FdC and 5-CldC are good substrates for both enzymes. The analogs present opportunities for Collateral Sensitivity for 5-azacytidine and gemcitabine resistant tumors.