S. Spiegelman

Purification of the DNA Polymerase of Avian Myeloblastosis Virus

Abstract DNA polymerase from avian myeloblastosis virus has been purified by a combination of column chromatography and gel filtration methods. The isolated enzyme sediments at approximately 6 S and consists of two subunits of molecular weights 110 000 and 69 000. It is free of RNA and DNA endonuclease activity. The enzyme possesses the RNA-, DNA-, and hybrid-directed polymerase activities found in the virion.

An overview of thymidine

This review summarizes a body of information suggesting that proper metabolic modulation with certain metabolites can sensitize tumor cells to antimetabolites, and others can de‐sensitize (i .e . protect) normal cells from the toxicity of antimetabolites. This new approach offers the possibility of increasing the selectivity of drug therapy, with the promise of a real advance in cancer chemotherapy. The metabolite thymidine (TdR), long used as a cell synchronizing agent, is known to exert this effect in vitro by metabolic modulation of a number of enzymes in the salvage pathway to DNA synthesis. Against this biochemical background, in vivo effects of TdR employed as an agent for cancer therapy are reviewed as follows: 1) TdR alone, and in combination with, 2) Methotrexate (MTX), or 3) 5‐Fluorouracil (FU), or 4) Cytosine arabinoside (ara‐C). TdR is shown in all instances either to protect against host toxicity (eg. MTX), or to potentiate the anti‐tumor effect (eg. FU and ara‐C). Findings are also presented that a sequential schedule of MTX prior to TdR prior to FU is important for the optimal therapeutic activity of these drugs. The biochemical basis for the MTX → FU augmentation is reportedly due to increased activation of FU by MTX (acting indirectly). On the basis of this biochemical insight, a completely different chemotherapeutic agent methyl‐mercaptopurine rihoside (MMPR) was substituted for MTX, resulting in a dramatic potentiation of anticancer activity. Metabolic modulation with still other metabolites (UR) and a hormone (testosterone) was demonstrated to protect from host toxicity due to certain anti‐cancer agents without offsetting anti‐tumor activity. The ability to prevent leukopenia by these means was particularly impressive. Clinical trials have been initiated with TdR alone, TdR + MTX, and TdR + FU; the available clinical data are summarized.

Potentiation of the anti-tumor activity of 5FU by thymidine and its correlation with the formation of (5FU) RNA

Evidence is presented with two murine tumor systems (CD8F1 mammary carcinoma and CD2F1 colon tumor 26). We question the thesis that the antitumor activity of 5‐fluorouracil (FU) is achieved solely by the inhibition of thymidylate synthetase by the derivative, fluorodeoxymonophosphate (FdUMP). The data described here are more consistent with the formation of FU‐containing RNA (FU‐RNA), an event which can adversely affect a variety of cellular mechanisms requiring RNA processing and function. These are not two mutually exclusive mechanisms. However, if the deleterious effect of FU‐RNA constitutes a significant quantitative component of the anti‐neoplastic activity of FU, the addition of thymidine would be expected to increase the antitumor activity of FU by the following three mechanisms: 1) protection of FU from catabolic degradation by saturation of the relevant enzymes with thymidine; 2) selective arrest by thymidine “feedback” of normal cells compared with malignant cells; 3) feedback repression of the FU anabolic pathways leading to deoxyderivatives, thus encouraging the entry of FU into RNA.

Conditions for the selective synthesis of DNA complementary to template RNA.

Abstract The effect of actinomycin D on the DNA synthesized by RNA-instructed polymerase (reverse transcriptase) has been examined. Preferentially, DNA complementary to viral RNA (i.e. DNA (−)) is synthesized at high antibiotic concentrations with either detergent-disrupted virions or purified avian myeloblastosis virus (AMV) polymerase and exogenous viral RNA templates. DNA-instructed DNA synthesis is severely inhibited under these conditions. Reactions with synthetic homopolymer templates indicate that deoxyguanosine residues are necessary for inhibition and that actinomycin D has no effect directly on the polymerase. The antibiotic distamycin A yields similar results to those obtained with actinomycin D, except that deoxythymidine residues are necessary for inhibition. The methods detailed here may be used to detect reverse transcriptase and to prepare purified single-stranded DNA transcripts complementary to a variety of RNA template molecules.

The purification of a gs antigen of the murine mammary tumor virus and its quantitation by radioimmunoassay

Abstract A method using Concanavalin (ConA) affinity chromatography has been developed for the purification of the mouse mammary tumor virus (MuMTV) gs antigens (gp52) and (p27). The procedure combines ConA chromatography and DEAE cellulose fractionation to yield the gs antigens in both high yield and sufficient purity for use in radioimmunoassay (RIA). The gp52 obtained has been iodinated with the [ 125 I]Bolton-Hunter reagent, chromatographed on Sephadex G-100, and utilized in the development of a blocking radio immunoassay (RIA). The sensitivity of this assay (0.5–1.0 ng) is superior to that achieved with whole particle assays. The procedures used for the isolation and iodination of the gp52 avoid the technical problem of incomplete displacement of [ 125 I]gp52 by native viral antigen. The resulting RIA provides a sensitive, reliable tool for studying the expression of the gp52 antigen and its relationship to the onset and progression of murine breast cancer.

Human cancer and animal viral oncology

DNA‐RNA hybridization was used to explore whether human neoplasias contain RNA molecules having sequence homologies to those of the RNA tumor viruses known to cause similar diseases in animals. The pattern of specific RNAs found in the human tumors showed a remarkable concordance with the predictions deducible from the animal systems. Thus human breast cancer contains RNA homologous only to that of the murine mammary tumor virus (MMTV). Human leukemias, sarcomas, and lymphomas (including Hodgkins and Burkitts) all contain RNA with sequence homology to the murine leukemia virus (RLV) and not to MMTV RNA. Finally, as in the case of the mouse, none of the human tumors examined contains RNA related in sequence to that of the avian myeloblastosis virus. The RNA detected in all of the human neoplasias was demonstrated to be of high molecular weight (1 × 107 daltons) and encapsulated with a reverse transcriptase in particles having densities between 1.16–1.19 g/ml. Further, the RNA of these human tumor particles was related in sequence to the murine viruses that cause the corresponding neoplasias in mice. Thus, four features diagnostic for the murine oncogenic viruses are satisfied by the particles found in the human cancers. Finally, it was shown by “recycling” experiments that the DNA from human leukemic cells and from lymphomatous tissue contained particle‐related sequences that could not be detected in normal DNA. This finding was further substantiated by studies with identical twins, in which it was shown that the leukemic twin contained particle‐related sequences that could not be detected in the leukocytes of his identical healthy sibling. These findings are inconsistent with hypotheses that require chromosomal transmission in the germ line of complete copies of the information required to produce malignancy and the associated virus particles.

Effect of Uridine on the Metabolism of 5-Fluorouracil in the CD8F1 Murine Mammary Carcinoma System

The effect of uridine on the incorporation of 5-fluorouracil into RNA and the inhibition of DNA synthesis by the FdUMP block of thymidylate synthetase was studied in the CD8F1 murine mammary carcinoma system. The administration of exogenous uridine resulted in about a one third reduction of 5-fluorouracil in RNA of tumor and normal tissues. However, unlike thymidine, uridine was unable to reverse the early, partial inhibition of DNA synthesis. The amount of fluorouridine nucleotides and (5-fluorouracil)RNA formed in various tissues correlates with the level of orotate phosphoribosyl transferase activity suggesting that the major pathway for activation of 5-fluorouracil to nucleotide form in these tissues is via phosphoribosyl transferase. Enzyme preparations from three different murine tumors convert about 15 times as much 5-fluorouracil to FUMP as they do uracil to UMP. In contrast, the ratio of FUMP to UMP formed in enzyme preparations from gut and bone marrow is lower, 2–6 fold. However, in none of these tissues was the in vitro conversion of 5-fluorouracil to FUMP or incorporation into RNA substantially inhibited by uracil. Examination of tumor, gut and bone marrow uridine nucleotide pools showed that the thymidine-uridine-5-fluorouracil schedule does increase uridine nucleotide pools. Thus, the reduction in 5-fluorouracil in RNA is probably not due to inhibition of the conversion of 5-fluorouracil to FUMP by uracil (derived from phosphorylase cleavage of uridine) but, rather, is probably due to the elevated levels of UTP. We conclude that the protection from 5-fluorouracil toxicity afforded by the addition of uridine is due to the reduction in 5-fluorouracil in RNA rather than by reversal of the FdUMP block on thymidylate synthetase.

The mechanism of Qβ replication: Sequence at the 5′ terminus of a 6-S RNA template

Abstract The infection of Escherichia coli by the RNA-containing bacteriophage Qβ results in the appearance of several distinctive RNA species. One of these species with a sedimentation coefficient of 6S serves as a template for the viral replicase and seems to possess an unusual secondary structure. The nucleotide sequence at the 5′ terminus of a cloned 6-S RNA was determined to be pppGpGpGpApUp.

Serological analysis of reverse transcriptase of the Mason-Pfizer monkey virus

Abstract Antiserum prepared against partially purified DNA polymerase from Mason-Pfizer monkey virus neutralized the endogenous DNA polymerase of that virus and of X381, an agent morphologically indistinguishable from MPMV, isolated from cultures prepared from the lactating mammary gland of a rhesus monkey. The antiserum did not inhibit the DNA polymerase activities of avian myeloblastosis virus, feline leukemia virus, Rauscher murine leukemia virus, Friend murine leukemia virus, murine mammary tumor virus, and simian sarcoma virus-1.

Diagnosis of primary breast carcinoma through immunohistochemical detection of antigen related to mouse mammary tumor virus in metastatic lesions: A report of two cases

Recent investigations have established that approximately half of human breast carcinomas contain an immunohistochemically detectable antigen which is cross‐reactive with the 52000‐dalton major glycoprotein (gp52) of the mouse mammary tumor virus (MMTV). This antigen can be localized in paraffin‐embedded sections of routinely fixed tissues using heterologous antibodies to gp52 or MMTV. This report describes two patients with metastatic carcinoma in axillary lymph nodes without any clinical evidence of a primary lesion in the breast or elsewhere. The localization of the gp52‐related antigen in paraffin‐embedded sections of both metastatic lesions suggested the presence of primary mammary carcinoma. In both instances, this suggestion was ultimately confirmed by the finding of primary lesions in which the gp52‐related antigen was also found.