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 [ 125 I]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 [ 3 H]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- 3 H]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. </jats:p

Preferential Lysis of Pancreatic B-Cells by Islet Cell Surface Antibodies

Sera from patients with insulin-dependent diabetes mellitus (IDDM) containing islet cell surface antibodies (ICSA) were studied for their capacity to lyse cultured rat islet cells. The uptake of ethidium bromide was used to identify lysed cells and immunofluorescent staining with antisera to insulin, glucagon, somatostatin, or pancreatic polypeptide was used to identify the different islet cell types (B-, A-, D-, and PP-cells, respectively). Our experiments showed that in the presence of complement, sera containing ICSA lysed 81% of the B-cells, but 10% or less of the A-, D-, and PP-cells. Normal control sera resulted in lysis of less than 4% of each of the islet cell types. The demonstration that ICSA are preferentially lytic for B-cells may be important in defining the role of these autoantibodies in the pathogenesis of IDDM, particularly since the B-cell mass in diabetics is markedly reduced relative to the other islet cell types.</jats:p

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 [(125)I]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 [(3)H]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-(3)H]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