Murine Mammary Tumor Virus RNA Research Articles

Early events in the stimulation of mammary tumor virus RNA synthesis by glucocorticoids. Novel assays of transcription rates.

Two procedures were devised to examine initial events in the glucocorticoid-regulated transcription of integrated murine mammary tumor virus (MTV) DNA sequences. First, cells in monolayer culture were exposed for brief periods to dexamethasone and then permeabilized in situ with digitonin to allow entry of 32P-labeled ribonucleoside triphosphates and measurement of transcription rates. The results revealed that the rate of MTV RNA synthesis is stimulated selectively by dexamethasone after an apparent lag of approximately equal to 1 min and is half-maximal by 8-9 min. Second, the time course of transcription elongation was monitored by quantitating in single strand nuclease protection assays the accumulation of RNA sequences from different regions of the 7.8-kilobase MTV transcription unit. These experiments suggested that the rate of MTV RNA elongation in the presence of hormone does not differ substantially from elongation rates calculated for other genes transcribed by RNA polymerase II. Moreover, dexamethasone increased in parallel and to the same extent the levels of RNA from various segments of the transcription unit, implying that transcripts are elongated across the MTV element at a constant rate. We conclude that glucocorticoids stimulate MTV transcription solely via a rapid and selective increase in the efficiency of transcription initiation.

Expression and demethylation of germinally-transmitted BALB/c mouse mammary tumor virus DNA in Abelson MuLV B-lymphoid cell lines.

Murine mammary tumor virus (MMTV) RNA expression, DNA organization and DNA demethylation were examined in BALB/c B-lymphoid cell lines produced by transformation with the Abelson murine leukemia virus (AbMuLV). The MMTV DNA sequences in AbMuLV B cell lines, based on restriction mapping with EcoRI, PstI, BglII, BamHI and SacI and molecular hybridization with cloned probes of the MMTV LTR, gag-pol or env gene regions, were identical to the germinally-transmitted MMTV DNA complement of BALB/c mice. Several AbMuLV B cell lines expressed MMTV poly(A+)-RNA at detectable levels. MMTV poly(A+)-RNA for the env gene, 3.8 kb, and the long terminally redundant (LTR) region, 1.7 kb, were detected in some AbMuLV B cell lines. MMTV DNA sequences in the AbMuLV B cell lines were at least partially sensitive to digestion by the methylation-sensitive restriction endonucleases HhaI and HpaII. HhaI-sensitive sites were present in Units I, II and III of the germinally-transmitted MMTV DNA and were localized specifically near the 5' end of the 5' and 3' LTRs of both Units II and III. HpaII-sensitive sites were localized near the 3' end of the 3' LTRs of Units II and III, and at a cellular site 2.1 kbp 5' to the 5' LTR. These observations demonstrate that the germ line MMTV DNA sequences of BALB/c mice are expressed in cells of B lymphocyte origin, and suggest a correlation between MMTV RNA expression and selective demethylation in the LTR regions of germinally-transmitted MMTV DNA sequences.

Glucocorticoids and chromosomal position modulate murine mammary tumor virus transcription by affecting efficiency of promoter utilization.

The rate of transcription of murine mammary tumor virus (MTV) sequences in MTV-infected rat hepatoma tissue culture cells is strongly affected by both glucocorticoid hormones and the chromosomal position of provirus integration. We have characterized MTV RNAs produced in J2.17 and M1.54, independent isolates containing, respectively, 1 and 10 proviruses integrated at distinct chromosomal loci. M1.54, but not J2.17, synthesized MTV RNA in the absence of glucocorticoids; the rate of hormone-stimulated viral gene transcription in M1.54 was 50- to 100-fold higher than in J2.17. In each case in which MTV genes were expressed (J2.17 induced, M1.54 basal and induced), the viral RNAs produced were indistinguishable. RNA blotting revealed accumulation of two transcripts, 7.8 and 3.8 kilobases; the latter was likely produced from the former by RNA splicing. Sites used for transcription initiation, polyadenylation, and splicing have been identified from the sizes of end-labeled hybridization probes protected from digestion with mung bean nuclease; the unique initiation and polyadenylation sites were both encoded within the MTV long-terminal-repeat sequence. The efficiencies of splicing and of utilization of the polyadenylation signal did not appear to vary as functions of chromosomal position or hormonal stimulation. Differences in rates of viral gene transcription were reflected in the differential accumulation of the 5'-terminal 136 nucleotides of MTV RNA. Thus, glucocorticoids and chromosomal position appeared to affect solely the efficiency of utilization of the MTV promoter, leaving unchanged the sites of initiation, splicing, and polyadenylation, as well as the efficiencies of the latter two processes.

Mammary tumor virus DNA contains sequences required for its hormone-regulated transcription

Glucocorticoids regulate the rate of transcription of integrated murine mammary tumor virus (MTV) genes in most clones of MTV-infected rat hepatoma tissue culture (HTC) cells. To determine whether hormonal regulation is mediated from flanking cellular sequences or rather from within the viral DNA, we analyzed the relative rates of trancription of MTV and adjacent HTC sequences in two lines of infected HTC cells, J2.15 and J2.17, each of which contains a single insertion of MTV DNA. In addition, we measured in uninfected HTC cells the transcriptional rates of the corresponding “preinsertion fragments,” which may be viewed as the equivalent sequences bearing deletions of MTV DNA; the two genomic segments into which integration occurred in the two lines are unrelated. Previous work showed that glucocorticoids induce the accumulation of MTV RNA in J2.17, whereas viral transcripts are not detected in J2.15. RNA pulse-labeling experiments indicate that glucocorticoids stimulate the rate of MTV gene transcription in J2.17 but not in J2.15; in contrast, no labeled RNA hybridizing to flanking sequences was detected either in the uninfected or in the infected cells. We conclude that the host site of integration of MTV need not be transcriptionally active or hormonally responsive to permit viral gene expression. Furthermore, these rate measurements indicate that glucocorticoid-stimulated MTV RNA synthesis in J2.17 does not reflect readthrough transcription from a regulated cellular promoter. This notion is independently supported by transcript mapping experiments showing that the 5′ terminus of nuclear MTV RNA is at a site on MTV DNA approximately 1.2 kb downstream from the host-viral junction. Thus our data are consistent with the presence of a hormone-responsive element within the provirus.

Induction of Endogenous Mammary Tumor Virus Expression During Hormonal Induction of Mammary Adenoacanthomas and Carcinomas of BALB/c Female Mice<xref ref-type="fn" rid="FN2">2</xref><xref ref-type="fn" rid="FN3">3</xref>

Treatment of BALB/c female mice with pituitary isografts, under conditions that established mammary hyperplasia in an anovulatory condition, enhanced mammary tumor development with prior onset of dysplastic foci in lobular parenchyma. Tumor onset began at 8 months (mean onset time, 18 mo); the 25-month incidence was 100%. Adenoacanthomatous tumors appeared first. Adenocarcinomas appeared only after more than 14 months of continuous hormone stimulation. Dysplastic and neoplastic changes occurred while blood levels of the three major mammotropic hormones were physiologic. Murine mammary tumor virus (MuMTV) p28 was detected in all tumors tested, independent of time of tumor appearance or tumor type, although keratinizing cells in adenoacanthomatous tumors did not contribute to MuMTV antigen expression. MuMTV gp52 was detected in only a small fraction of cells in a few tumors. MuMTV RNA contents were above normal in all tumors tested. Neither MuMTV structural antigens nor RNA was induced in normal glands by the same hormone treatment that ultimately resulted in dysplasia and tumor formation and elevated levels of these viral markers in neoplasms. The MuMTV RNA in all hormone-induced tumors was readily distinguishable in base sequence from standard MuMTV RNA but indistinguishable from MuMTV RNA recovered from lactating mammary glands of BALB/c females carrying only endogenous MuMTV proviruses, suggesting that endogenous MuMTV RNA sequences were induced in hormone-induced neoplasms. RNA indistinguishable from MuMTV sequences present in hormone-induced primary tumors was also detected in multiple genomic equivalents in two independently derived hormone-induced premalignant alveolar hyperplasias. MuMTV p28 was detected, but gp52 was not. The same hormone stimulus that generated 100% tumors in normal gland greatly accelerated tumor development in premalignant hyperplasias but did not amplify MuMTV RNA or antigens in either hyperplasias or the tumors derived from them. B-type virions were not detected in these tissues, in either hypophyseal implant-stimulated or virgin hosts. Cell-free virions were not detected in culture. These data suggest that the replication of MuMTV induced in hormone-induced neoplasms is defective. Details of its expression suggest that if involved in events leading to tumors, its involvement is insufficient cause for those tumors.

Quantitative of murine mammary tumor virus-related RNA in mammary tissues of low- and high-mammary-tumor-incidence mouse strains.

Lactating mammary glands and hormonally induced mammary tumors of BALB/c mice from three geographically separated breeding colonies were examined by molecular hybridization, using murine mammary tumor virus (MuMTV) cDNA representing the entire viral genome to determine the amount of MuMTV-related RNA expressed in these tissues. The RNA extracted from these tissues by the classical sodium dodecyl sulfate-pronase, phenol-chloroform procedure (method 1) contained barely detectable levels of MuMTV-related sequences. In contrast, both normal lactating mammary glands and hormonally induced mammary tumors of these mice were found to contain approximately one to two copies of the MuMTV genome per cell by using a new procedure in which the RNA was extracted with guanidine derivatives (method 2). No significant differences in the MuMTV-related RNA content of the BALB/c mammary tissues were observed regardless of their colony of origin. Our results suggest that expression of MuMTV RNA does not change in BALB/c mammary glands during transformation to a malignant state and that MuMTV expression does not play a role in tumorigenesis in these mice. In view of the increased recovery of MuMTV-related RNA from BALB/c mice with method 2, we compared the level of MuMTV RNA expression in lactating mammary glands and mammary tumors of other mouse strains, including C57BL/6 and RIII, using both extraction methods. Yields of MuMTV-related RNA from mammary tissues increased by as much as 35- to 40-fold, using method 2 as compared with method 1. Therefore method 2, involving guanidine derivatives, appears to be method of choice for MuMTV-related RNA extraction from the mammary tissues of certain strains of mice, particularly those expressing relatively low levels of MuMTV RNA.

Lack of induction of murine mammary tumor virus expression in cultured mammary glands treated with chemical carcinogens

AbstractChemical carcinogens have previously been found to bring about an epithelial transformation of mouse mammary glands in whole‐organ culture, as evidenced by the escape of lobuloalveoli from the normal hormonal controls of mammary development, and by glandular hyperplasia, dysplasia and metaplasia. In order to assess whether a general induction of endogenous murine mammary tumor virus (MuMTV) genes is associated with the chemical transformation process, we assayed steady‐state levels of MuMTV RNA and protein after treatment of the cultured mammary glands with the carcinogens, 7,12‐dimethylbenz[a]anthracene and N‐2‐fluorenylacetamide. Viral RNA was measured by hybridization with a representative MuMTV‐complementary DNA probe, and viral protein was analyzed by a group‐specific radioimmunoassay for the major MuMTV envelope glycoprotein, gp52. In control glands not treated with carcinogen, there were extremely low levels of MuMTV‐specific RNA in the cultured mammary glands originating from BALB/c mice, and between 10‐ and 50‐fold higher levels in glands from C57BL/6 and C3H mice. Among the untreated glands, only those from C3H mice contained a detectable level of gp52. This order of MuMTV gene expression in the cultured glands of the three strains of mice is consistent with that observed in vivo. Treatment of the cultured mammary glands of these three strains of mice with either of the two chemical carcinogens resulted in all cases in no elevation in the level of either MuMTV RNA or protein. Thus, our studies indicate that the transformation of the cultured mammary glands by the two chemical carcinogens occurs by a process that apparently does not involve a general induction of endogenous MuMTV gene expression.

Reevaluation of the Effect of Mouse Mammary Tumor Virus Infection on the BALB/c Mouse Hyperplastic Outgrowth<xref ref-type="fn" rid="FN2">2</xref><xref ref-type="fn" rid="FN3">3</xref>

The BALB/c (C-) mouse hyperplastic outgrowth line (D1) was used to study murine mammary tumor virus (MuMTV) expression in both D1 and tumors derived from D1. D1 was transplanted into virus-infected BALB/cfC3H (C+) and virus-uninfected C- animals. In duplicate studies, tumor incidence was the same in both groups. However, the tumor latency period was longer for D1 transplanted into C+ mice (D1/C+) than for D1 transplanted into C- mice (D1/C-). MuMTV was detected in 85% of D1/C+ outgrowths and in 29% of D1/C+ tumors but was never detected in D1/C- outgrowths or tumors. D1/C- outgrowth and tumors and most of the D1/C+ tissues expressed little or no MuMTV RNA. Some D1/C+ tumors expressed substantial levels of MuMTV RNA. These same tumors also had MuMTV antigen and contained the exogenously acquired C3H-MuMTV provirus in the cellular DNA as shown by DNA fragment patterns following cleavage with Pst I and Eco RI endonucleases. D1/C+ tumors containing no viral RNA were also antigen-negative and lacked the acquired C3H provirus. These studies indicate that D1 has substantially changed in its incidence and in its response to MuMTV. MuMTV infection was not tumorigenic in the traditional sense, a finding that has led to a reevaluation of our current models of virus-host relationships and the biology of precancerous conditions.

Organization and expression of endogenous murine mammary tumor virus genes in mice congenic at the H-2 complex

The organization and expression of endogenous murine mammary tumor virus (MuMTV) genetic information was examined in a number of mouse strains Congenic at the major histocompatibility complex (MHC). Analysis of restriction endonuclease-generated fragments of cellular DNA revealed no differences in the integration sites ( EcoRI)or internal cleavage sites ( PstI) of MuMTV proviruses in any of the (C7BL/10 (B10) Congenic strains tested. Similar studies indicated that the proviruses of BALB/c and BALB.B mice are indistinguishable, and that the restriction endonuclease-generated fragments of BALB/c mice are similar to those of the B10 strain. These proviruses behave as stable genetic elements which have been well conserved during the derivation of the Congenic strains. Also, since none of the characteristic MuMTV proviruses of the donor strains could be identified in the Congenic mice, they do not seem to be closely linked to the H-2 complex. When concentrations of viral RNA in lactating mammary glands (LMGs) were measured by molecular hybridization kinetics, comparable levels of MuMTV RNA were found in all the B10 Congenic mice; considerably less viral RNA was detected in mice with a BALB/c background. This viral RNA in mammary gland cells was not translated into detectable gp49, the major MuMTV glycoprotein. These results indicate that the differences observed in susceptibility to mammary tumorigenesis induced in H-2 Congenic mice by exogenous MuMTV are not influenced by the organization or expression of endogenous MuMTV proviral genes.

Hormonal regulation of murine mammary tumor virus RNA expression during mammary tumorigenesis in BALB/c mice

The effects of glucocorticoids and prolactin on murine mammary tumor virus (MuMTV) RNA expression in preneoplastic outgrowth lines and mammary tumors in BALB/c mice were investigated. Hyperplastic alveolar nodules (HAN) and a ductal hyperplasia (DH) are induced in virgin BALB/c mice by prolonged hormonal stimulation or treatment with 7,12-dimethylbenz(a)anthracene or both. Mice bearing HAN or DH outgrowth lines and mammary tumors that arose from the outgrowth lines were treated with glucocorticoids or prolactin. MuMTV RNA was quantitated by hybridization with a representative complementary DNA probe specific for MuMTV RNA. Prolactin treatment did not increase MuMTV RNA in the BALB/c HAN or DH outgrowth lines or tumors. MuMTV RNA increased after glucocorticoid treatment in the C3, C4, and C5 HAN outgrowth lines and in tumors that arose from the D1, D2, C4, and C5 HAN and CD8 DH outgrowth lines. No increase in MuMTV RNA with glucocorticoid treatment was observed in the D1 or D2 HAN outgrowth line, in the CD8 DH outgrowth lines, and in tumors that arose from the C3 HAN outgrowth line. The ability of glucocorticoids to stimulate MuMTV expression was specific since the response was dose dependent and specific for glucocorticoid hormones. Glucocorticoid treatment did not increase the level of type C viral RNA in the majority of hormone- or 7,12-dimethylbenz(a)anthracene-induced HAN outgrowth lines or tumors. These observations suggested that glucocorticoids may influence MuMTV expression during mammary tumorigenesis in BALB/c mice.

Effect of dexamethasone on expression of endogenous mouse mammary tumor virus sequences in BALB/c tumor cell lines

BALB/c mammary tumor cell lines which contain only endogenous murine mammary tumor virus (MMTV) sequences respond to dexamethasone (DXS) treatment with minimal (approximately 2-fold) increases in MMTV RNA. This is in marked contrast to the 10− to 20-fold increases observed with cell lines harboring exogenous MMTV variants. Comparison of hybridization results obtained with two complementary DNA probes representative of either the entire MMTV RNA genome or its poly(A)-adjacent sequences suggests that the DXS response of BALB/c lines is also qualitatively different from that of exogenous MMTV-producer cell lines. Thermal stability studies suggested a 2–3% divergence between the RNA sequences of endogenous BALB/c and exogenous C3H viruses, with the 3′-end of the viral RNA appearing to be conserved relative to the rest of the genome.

Murine Mammary Tumor Virus Expression During Mammary Tumorigenesis in BALB/c Mice

Steady-state levels of murine mammary tumor virus (MuMTV) RNA were quantitated during mammary tumorigenesis in BALB/c mice by molecular hybridization with a representative MuMTV complementary DNA (cDNA) probe. Hyperplastic alveolar nodule (HAN) lines are preneoplastic mammary lesions that were induced in BALB/c mice by hormones alone or in combination with 7,12-dimethylbenz(a)anthracene and give rise to mammary tumors. The hormone-induced HAN lines D1 and D2 contained detectable amounts of hybridizable MuMTV sequences. MuMTV RNA sequences were also observed in five of the six transplanted BALB/c mammary tumors that were examined. Similar levels of hybridizable MuMTV RNA were observed between the D1 or D2 HAN line and mammary tumors derived from each HAN line. The D2 HAN line as well as D2, C4, and CD8 mammary tumors accumulated RNA that was apparently homologous to most of the MuMTV genome. Thermal denaturation of hybrids indicated extensive sequence homology between the MuMTV cDNA and hybridizable RNA in the BALB/c HAN lines and mammary tumors. A low level of type C viral RNA was observed in the BALB/c HAN lines and most mammary tumors by molecular hybridization with a cDNA to Moloney murine leukemia virus. These data demonstrate that MuMTV sequences are frequently expressed in hormone-induced BALB/c HAN lines and mammary tumors derived from HAN lines or ductal hyperplasias induced in BALB/c mice by hormones and/or a chemical carcinogen. The transition from the preneoplastic to the neoplastic state in BALB/c mice does not appear to be due to a change in the steady-state levels of MuMTV RNA since the hormone-induced HAN lines and mammary tumors had similar levels of hybridizable MuMTV RNA.

Murine mammary tumor virus (MuMTV) infection of an epithelial cell line established from C57BL/6 mouse mammary glands

An epithelioid cell line derived from the mammary glands of a C57BL/6 mouse and designated C57MG cell line was found to be susceptible to infection by routine mammary tumor virus (MuMTV). Although uninfected C57MG cells contain endogenous MuMTV-related DNA sequences, no RNA sequences homologous to MuMTV were detectable, even after treatment with the glucocorticoid, dexamethasone. However, after infection with MuMTV, these cells acquire additional MuMTV DNA, and viral RNA and proteins were readily detectable. Most, if not all, of the additional MuMTV DNA in infected C57MG cells appeared to be integrated. Synthesis of viral RNA and protein, and the release of virions into culture fluid by infected C57MG cells, was stimulated by incorporation of dexamethasone in the growth medium. The efficiency of infection by MuMTV in C57MG cells was similar to that in nonmurine cells. There was a direct relationship between multiplicity of infection (m.o.i.) and the average number of MuMTV RNA molecules detected per cell 5 weeks after infection. However, even at the highest m.o.i. used (4 × 10 5 virions/cell), a plateau in the average number of viral RNA molecules per cell was not achieved. The origin of MuMTV synthesized by infected C57MG cells, whether it is the progeny of infecting MuMTV or of the endogenous C57BL/6 MuMTV or of both, remains undetermined. We have been unable to detect any morphological or growth pattern changes in C57MG cells following infection with MuMTV.

Electrophoretic analysis of the molecular weight of murine mammary tumor virus RNA

Molecular weight determinations of native and subunit RNAs of murine mammary tumor virus (MuMTV), a type B oncornavirus, were performed by polyacrylamide gel electrophoresis and compared with molecular weights of well-characterized avian cellular RNAs and tobacco mosaic virus RNA. From extrapolations of semilog plots of the molecular weights of the standard RNAs versus relative electrophoretic mobilities and Ferguson plots, the subunit and native RNAs of MuMTV were found to possess molecular weights of 2.93 X 10(6) and 6.45 X 10(6), respectively. These data support the assumption that two subunit molecules comprise the native RNA of MuMTV.

ChemInform Abstract: MOLECULAR APPROACH TO THE ETIOLOGY OF HUMAN CANCER

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 RNA's 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. Human leukemias, sarcomas, and lymphomas (including Hodgkin's and Burkitt's lymphomas) all contain RNA with sequence homology to the murine leukemia virus and not to murine mammary tumor virus RNA. Finally, as in the case of the mouse, none of the human tumors examined contains RNA 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 and 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.

Molecular Approach to the Etiology of Human Cancer

Abstract 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 RNA's 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. Human leukemias, sarcomas, and lymphomas (including Hodgkin's and Burkitt's lymphomas) all contain RNA with sequence homology to the murine leukemia virus and not to murine mammary tumor virus RNA. Finally, as in the case of the mouse, none of the human tumors examined contains RNA 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 and 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.

Mammary tumor virus induction by glucocorticoids. Characterization of specific transcriptional regulation.

Dexamethasone (1,4-pregnadiene-9-fluor-16alpha-methyl-11beta,17alpha,21-triol-3,20-dione), a potent synthetic glucocorticoid, stimulates mouse mammary tumor virus expression 10- to 20-fold in tissue culture cells. This hormone effect was observed at concentrations as low as 1 times 10-10 M and was maximal at 10-7 to 10-8 M. The time course of induction indicated that detectable increases in extracellular viral DNA polymerase were first noted 18 to 24 hours following the addition of dexamethasone, and cells produced the highest polymerase levels at the time monolayers approached confluence. Steroid responsiveness was associated with specific increases in type B murine mammary tumor virus structural polypeptide (gp52(sl) expression and murine mammary tumor virus RNA that quantitatively paralleled the increase in extracellular virus production as measured by electron microscopy and supernatant RNA-dependent DNA polymerase activity. Another virally transformed murine cell line, KA 31, did not contain detectable levels of murine mammary tumor virus gp52(sl) or RNA before or after dexamethasone stimulation; thus induction was noted only in murine cells with pre-existing murine mammary tumor virus expression. No increase in basal levels of type C murine leukemia viral proteins or RNA was detected in dexamethasone-treated mammary cell lines which were producing increased levels of murine mammary tumor virus. Therefore, increases in murine mammary tumor virus gene products are specific for murine mammary tumor virus DNA sequences under these conditions.

Glucocorticoid-receptor interaction and induction of murine mammary tumor virus.

The relationship between the cellular uptake of glucocorticoid hormones, the binding of these hormones to specific in vitro receptors, and the induction of mouse mammary tumor viruses in an established mouse mammary tumor cell line was highly correlated. These results suggest that the induction of mouse mammary tumor virus by glucocorticoid hormones is a physiological process acting through a mechanism of high affinity, saturable steroid-receptors. A temperature-sensitive or salt-dependent step following glucocorticoid-receptor interaction was required for nuclear uptake of the steroid. Induction studies with different adrenocorticoids indicate that the synthetic glucocorticoid, dexamethasone (1,4-pregnadiene-9-fluor-16alpha-methyl-11beta,17alpha,21-triol-3,20-dione), is the most potent inducer of mouse mammary tumor viruses and all steroids which caused significant induction were glucocorticoids. Other glucocorticoids appear to stimulate murine mammary tumor virus production by a mechanism similar to that of dexamethasone; for example, corticosterone competes with dexamethasone for binding to the glucocorticoid receptor and blocks the uptake of dexamethasone into cells. Progesterone also blocks the cellular uptake of dexamethasone and can bind to the glucocorticoid receptor at low concentrations (10-7 to 10-8 M) but progesterone does not consistently induce virus at hormone concentrations even as high as 10-4 M. Thus, in this system, binding to a cytoplasmic receptor is necessary but not sufficient for induction by glucocorticoids. Estrogens and androgens interfere with receptor binding and cellular uptake of dexamethasone but only at much higher concentration (10-4 M) than progesterone, and do not induce mammary tumor virus production. Although there was a positive correlation between steroid structure, binding, and biologic induction, other factors clearly affect the physiological manifestations of steroid actions. Mouse cells with comparable cytoplasmic receptor levels and comparable nuclear uptake differed absolutely in their degree of murine mammary tumor virus induction following hormone treatment. Although all mouse cells examined contain comparable levels of murine mammary tumor virus DNA, only cells producing constitutive levels of murine mammary tumor virus RNA could be induced to higher levels by a variety of glucocorticoids.

Sequences related to the RNA tumor viruses in the RNA and DNA of human leukemias and lymphomas.

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 Hodgkin's and Burkitt's) 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 contain RNA related in sequence to that of the avian myeloblastosis virus (AMV). The RNA detected in all of the human neoplasias was demonstrated to be of high molecular weight (1 times 10(7) 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, 4 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.