GR Mammary Tumor Research Articles

Quantitative flow cytometry of mouse mammary tumor virus envelope glycoprotein (gp52): alternative measures of hormone-mediated change in a viral cell surface antigen

An immunofluorescence procedure with C3H mouse mammary tumor cells (Mm5mt/cl) has incorporated flow cytometry to provide a fluorescence-based measurement of changes in the mouse mammary tumor virus (MMTV) cell surface glycoprotein (gp52). A comparison of mean channel fluorescence intensity (Δ mean) of cell populations stained with immune sera and NRS permitted a gp52-specific signal to be measured for controls and cells treated with 10 −6 M dexamethasone (Dex). Three different methods have been developed to quantitatively compare gp52-related fluorescence on control and hormone-treated cells. First, Δ mean, measured as a gp52-specific difference in channel number was 169–209 for control cells and 299–341 for Dex-treated cells. These fluorescence measurements with 4 different sera demonstrated gp52-specific increases due to Dex treatment of 141, 130, 143, and 115 channels. A second method of gp52 quantitation determined the percentage shift in staining populations over NRS and specified channel intensity markers. Dex treatment resulted in a 6.9 to 32.4% shift over channel 508 (NRS marker) and a more marked shift of 45.5 to 49.2% over channel 676 (control cell marker). A third methodology utilized fluorescein bead standards to calculate molecules of equivalent soluble fluorescein (MESF). These MESF determinations permitted hormonal effects to be measured as fold increases over controls. Dex induction of gp52 for C3H and GR mammary tumor cells ranged from 1.5 to 9.1 fold increases. Alternative steroid treatments and antibody directed against the internal cytoplasmic MMTV P27 provided negative controls for measurements of changing gp52 levels.

Isolation of a pathogenic clone of mouse mammary tumor virus

Exogenous mouse mammary tumor virus (MMTV) was cloned from a GR mammary tumor. Clone lambda GRT39 contained a full-length integrated MMTV(GR) provirus and both 5' and 3' host flanking DNA. The lambda GRT39 provirus had no apparent structural changes associated with cloning and retained the exogenous MMTV gag gene poison sequence. When introduced into rat mammary adenocarcinoma LA7 cells, the lambda GRT39 provirus was fully expressed. lambda GRT39-transfected LA7 cells made MMTV RNA, had gp52 SU protein on the cell surface, and produced B-type retrovirus particles characteristic of MMTV. Mammary tumors developed in hormone-stimulated BALB/c females injected with MMTV from lambda GRT39-transfected LA7 cells [MMTV (lambda GRT39)]. The tumors had new, clonally integrated copies of the MMTV(lambda GRT39) provirus and were expressing MMTV antigen. These data indicate that the lambda GRT39 provirus is biologically active and pathogenic.

Different int-1 region DNA rearrangements within different zones of a single mouse mammary tumor

Fragments were taken from separate parts of hormone-dependent (HD) primary GR mouse mammary tumors and serially transplanted in estrone plus progesterone treated or hormonally untreated castrated mice. The transplants were examined with respect to int-1 DNA rearrangement, proviral integrations of the murine mammary tumor virus (MMTV), and estrogen and progesterone receptor content. One of the fragments ( b) taken from the primary tumor of line TSI 96 produced transplants that showed int-1 rearrangement in one allele and also MMTV proviral integrations not at the int-1 gene, whereas transplants from another fragment ( a) only had the normal germ-line int-1 arrangement and no extra MMTV provirus. These respective genotypes were retained when the tumors became hormonally independent during further transplantations. The results indicate that int-1 rearrangement was not present in the originally transformed cell but occurred in a HD cell during growth of the tumor. Furthermore they indicate that loss of hormonal dependence in GR mammary tumors is due to a mutational event, unrelated to int-1 rearrangement.

Hormonal regulation of mouse mammary tumor growth

In view of reports that human breast cancer cells secrete growth factors that can replace estradiol in sustaining tumor growth [1], we have investigated whether hormone independent (HI) GR mouse mammary tumors can sustain growth of estrogen-depleted hormone dependent (HD) tumors. HD GR mammary tumor TS1 106 was grafted subcutaneously in the right flank of estrone plus progesterone treated castrated (020 × GR)F 1 mice. After 2 weeks the estrone treatment was stopped and the mice received 50,100 or 150 mg HI GR mammary tumor TS1 104 in the left flank. However, the regression of the HD tumor due to estrone depletion was not prevented or retarded by the HI grafts. In other experiments we investigated integrations of mouse mammary tumor virus (MMTV) proviral DNA in the DNA of GR mammary tumors. We could demonstrate the presence of two cell populations in tumor TS196, both HD but differing in MMTV DNA integration events. Our data indicate that exogenous integrations of MMTV proviruses can take place in mouse mammary tumor DNA without loss of hormone dependency of the tumors. Like in GR/Mtv-2 + mice, mammary tumor transplants differing in MMTV proviral integrations are also observed in 020/Mtv-2 + mice.

Detection of type-specific antigenic determinants of murine mammary tumor viruses with cell-mediated immune assays.

Exposure of lymphoid cells from normal and mammary tumor-bearing Balb/cCrgl mice to MMTV antigens result in positive responses in blastogenic transformation and migration inhibition reactions. The levels of these responses appear to depend on the murine strain source of the viral preparations. Enhanced reactivities were observed in cultures exposed to purified MMTV from culture fluids of mammary tumor cells of spontaneous origin in the C3H strain when compared to purified MMTV derived from milk of mice from the RIII strain. To ensure the virus relatedness of the differences observed, MMTVs obtained from a common source, i.e., the Crandall feline embryo kidney cell line, were employed in blastogenesis assays. Parallel testing of feline cell-derived C3H, RIII, and GR mammary tumor viruses resulted in different levels of [3H]thymidine incorporation by spleen cells from Balb/cCrgl mice over a range of viral concentrations. These results indicate that it is possible to detect various types of MMTV variants with cell-mediated immune assays. The diverse functional reactivities elicited by these viruses may be related to the differences observed in their bioactivities as determined by incidence and development of mammary tumors and the potential of causing metastases.

Dexamethasone induction of the intracellular RNAs of mouse mammary tumor virus.

We have studied the kinetics of dexamethasone induction of mouse mammary tumor virus (MMTV) RNAs and proteins in virus-infected rat XC cells and GR mouse mammary tumor cells. A detectable increase in viral RNA in infected XC cells was present within 10 min after hormone addition, and half-maximal induction was achieved in less than 2 h. The increase in viral RNA concentration was apparent first in nuclear RNA and later in the cytoplasm. Within the first 15 min of induction, only genome-sized RNA (35S, 7.8 kilobases) was present in augmented amounts, whereas the major subgenomic RNA (24S, 3.8 kilobases) did not appear until at least 30 to 60 min postinduction. The sequential appearance of these RNAs, the probable mRNA's for the gag and env proteins, paralleled the order of appearance of the gag and env proteins, respectively, after hormone treatment. An additional species of viral RNA (20S, 2.5 kilobases) was detected during these induction experiments, but the role of this RNA is not known. Both subgenomic RNAs contain sequences derived from both the 5' and 3' termini of genomic RNA and are presumably spliced. After dexamethasone induction of infected XC cells, we detected two smaller env-related proteins which were not found in full hormone induction. The functional role of these smaller proteins is not known. A previously reported smaller species of RNA (13S, 1.0 kilobase) did not appear to be induced and was shown to be cellular rather than viral in origin. In the fully induced infected XC and GR mammary tumor cells, the only viral RNAs present were the 35S and 24S RNAs. In addition, mammary tumors contained only these two viral RNAs. Thus, tumor cells appear to contain only the viral RNAs which direct the synthesis of the gag, pol, and env proteins of the virion.

Mammary tumors from gr mice contain more than one population of mouse mammary tumor virus-infected cells

Spontaneous mammary tumors in the GR mouse strain contain several acquired copies of mouse mammary tumor virus (MMTV) DNA that are not present in normal organ DNA and that are detectable by restriction endonuclease analysis and the Southern blotting procedure. Hormone-responsive and -independent cell populations were selected from spontaneous GR mammary tumors by grafting the tumors in castrated male GR mice in the presence and absence of female sex hormones. Analysis of the acquired MMTV DNA copies revealed differences between hormone-responsive and -independent cells derived from the same tumor; however, specific MMTV DNA fragments could not necessarily be correlated with the hormone responsiveness of the tumor. In some cases more than one proviral pattern could be detected for both hormone-responsive and hormone-independent cells. These results suggest that spontaneous GR mammary tumors are made up of more than one population of hormone-responsive and -independent cells which can be distinguished by their MMTV-specific proviral restriction fragments.

Characterization of endogenous and exogenous mouse mammary tumor virus proviral DNA with site-specific molecular clones.

Restriction fragments of the mouse mammary tumor virus (MMTV) proviral DNA were obtained by molecular cloning procedures. A 4-kilobase fragment delimited by two PstI sites was isolated from unintegrated, linear MMTV DNA and amplified in the pBr322 plasmid vector. EcoRI fragments of proviral DNA, integrated into the genome of a GR mammary tumor cell line, were isolated as lambda recombinant molecules. Five different recombinant phages which contained the 3' region of the MMTV proviral DNA and adjacent host DNA sequences were isolated. Heteroduplex analysis and S1 nuclease digestion suggested that there is no extensive sequence homology in the host DNA flanking the different proviral genes. The cloned DNA was fractionated into site-specific restriction fragments which served as molecular probes in the analysis of the endogenous MMTV proviral copies of C3H, GR, BALB/c, and feral mice. This allowed the correlation of MMTV-specific EcoRI fragments obtained from genomic DNA of these strains with the 5' and 3' ends of the proviral gene. Restriction fragments of two clones which contained the proviral sequences adjacent to the flanking host DNA as well as 1 to 2 kilobases of host DNA were used as hybridization probes, and the results allow the following conclusions: the proviral DNA of both clones contains nucleotide sequences complementary to the 5' and 3' ends of proviral DNA; and the host DNA flanking one clone belongs to the unique class of genomic DNA, whereas the DNA flanking the second clone is reiterated at least 15 times within the mouse genome.

Selective amplification of mouse mammary tumor virus in mammary tumors of GR mice

DNAs extracted from the mammary tumors of GR mice were analyzed for mouse mammary tumor virus proviral sequences by the restriction enzyme-Southern blot procedure. The tumor DNAs contain more proviral copies of mouse mammary tumor virus than DNA from a nonmalignant tissue. The degree of proviral amplification is small (ca. one to five additional copies) and appears to be variable from tumor to tumor. The restriction patterns of the amplified proviral sequences suggest a clonal origin for the tumor mass. In addition, the restriction patterns observed after digestion with the enzymes BglII and SacI indicate that only one of the proviruses endogenous to GR mice is amplified. The amplified provirus found in GR mammary tumors is identical to the provirus that is missing in GR-Mtv-2- mice, a congenic line exhibiting a low mammary tumor incidence.

Identification and partial characterization of an endogenous form of mouse mammary tumor virus that is transcribed into the virion-associated RNA genome.

Restriction mapping demonstrated the presence of several distinct proviral forms of mouse mammary tumor virus in the genome of GR mice. One of these proviruses (GR-MTV-2) was highly amplified in GR 3A cells, a cell line derived from a GR mammary tumor. By the criterion of restriction mapping, the amplified GR-MTV-2 provirus found in GR 3A cells was identical to the provirus found in M1.19 cells, a rat cell line infected with virions obtained from GR 3A culture fluid. This result clearly implies that the GR-MTV-2 provirus in GR 3A cells was transcribed into the virion-associated viral RNA genome. Cleavage of either GR 3A or M1.19 cell DNAs with the restriction enzyme Bg1 II gave rise to a 2.6 x 10(6) dalton GR-MTV-2 proviral fragment (ca. 45% of the viral genome). This fragment was isolated and mapped with thirteen restriction enzymes.

Gene order of mouse mammary tumor virus precursor polyproteins and their interaction leading to the formation of a virus

Mouse mammary tumor virus (MMTV) proteins are synthesized as two major precursor polyproteins; gPr75 env containing gp52 and gp36, and Pr75 gag containing p27, pp20, p14, and p10. The gene order for gPr75 env has been previously shown to be H 2N-gp52-gp36-COOH (Schochetman, et al., 1977). gag polyproteins undergo intracellular cleavage in cat cells infected with MMTV and GR mammary tumor cells. Based on immunoprecipitation studies with antisera against intermediate MMTV cleavage products we now report the gene order for Pr75 gag is H 2N-p10-pp20-p27-p14-COOH. These results were further substantiated by analyzing the binding to ssDNA of the intermediate cleavage products which contain p14. To analyze the interaction of MMTV proteins with the cell membrane leading to budding of a virus particle, we used (i) lactoperoxidase-catalyzed iodination of MMTV cell surface proteins, (ii) galactose oxidase-catalyzed radiolabeling of carbohydrates on cell surface MMTV glycoproteins, (iii) serum cytotoxicity based on [ 51Cr] release with monospecific MMTV antisera, and (iv) membrane immunofluorescence with monospecific MMTV antisera. Analysis of 125I-labeled MMTV cell surface antigens by immune precipitation with MMTV anti-gp52, gp36, p27, p14, and p10 sera followed by SDS-PAGE revealed only 125I-gp52. In contrast, cell surface glycoprotein labeling revealed [ 3H]gp52 and [ 3H]gp36, indicating that, although the protein portion of gp36 was buried, some carbohydrate regions were exposed. EDTA treatment of cells to alter cell membranes prior to iodination resulted in the labeling of both Pr75 gag and gp52 but not gPr75 env . Furthermore, anti-p10 but not anti-p27 serum was cytotoxic against EDTA-treated cells. Similar results were obtained when the same antisera were tested by membrane immunofluorescence, ruling out the possibility that anti-p27 serum was not cytotoxic because it was unable to fix complement. These results show that Pr75 gag molecules, presumably as MMTV cores, interact with cell membrane sites containing gp52 and gp36 via the hydrophobic p10 portion of the molecule.

Infection of cultured rat hepatoma cells by mouse mammary tumor virus

A continuous line of buffalo rat hepatoma (HTC) cells has been successfully infected with mouse mammary tumor virus (MMTV) produced by the GR mammary tumor cell line. Uniform infection required initial exposure of the HTC cells to >10 5 MMTV particles per cell. The resultant chronically infected cell population was found to have stably acquired 20–30 copies of MMTV DNA. The infected cells contain viral RNA and express viral antigens; however, very few MMTV particles are released into the culture medium. In spite of the biochemical evidence for infection, we have not detected any alterations in the morphology or growth properties of the infected HTC cells. As is the case in mammary tumor cells, the intracellular concentration of viral RNA is strongly stimulated (50–150 fold) by the synthetic glucocorticoid, dexamethasone. Thus it appears that the mechanisms by which glucocorticoids regulate MMTV gene expression in mouse cells are maintained when this virus infects nonmurine cells.