GR Mouse Mammary Tumors Research Articles

High prevalence of mouse mammary tumor virus-like gene sequences in breast cancer samples of Iranian women

ABSTRACTMouse mammary tumor virus (MMTV) is considered to be responsible for breast tumor development in mice. In several instances sequences homologous to the genome of this virus have been reported in human breast cancer samples.Here we aimed to evaluate MMTV involvement in human breast cancer development. DNA was extracted from 118 formalin fixed and paraffin embedded malignant (n = 59) and benign (n = 59) breast lesions. Using two sets of outer and inner MMTV-like envelope specific primers, in a nested PCR setup, MMTV genome was detected in 19 samples out of 59 (%32.2) cases of breast carcinomas, while in non-malignant breast tissue samples, only 3 samples out of 59 (%5) were positive. The difference was statistically highly significant (p < 0.001). Alignment of PCR amplified sequences with BR6, GR and C3H mouse mammary tumor virus strains emerged to be around 99% identical which is indicative of tumor tissue infection by an exogenous mouse MMTV genome.

Tumor growth inhibition by protein A and non-protein A containing Staphylococcus aureus in a mouse mammary carcinoma model.

Reinfusion of tumor-bearer plasma after absorption with killed Staphylococcus aureus strain Cowan I may be followed by inhibition or even acute necrosis of animal and human tumors. The effect has been attributed to protein A produced in large amounts by this staphylococcus. We have examined the effect upon the growth of a transplanted GR mouse mammary tumor of intraperitoneal inoculation of three strains of S. aureus characterized by being either protein A-rich or protein A-free. A significant tumor growth inhibition was found with all three strains of S. aureus. Serum levels of IgG1, IgG2 and IgM were found to be substantially increased. Crossed immunoelectrophoresis revealed increased numbers and titres of precipitins against staphylococcal antigens. It is concluded that staphylococcal moieties other than protein A may be involved in the tumor growth inhibition. The possibility and role of complement activation by protein A-like molecules through the alternative F(ab)2 reactivity is discussed.

Phosphorylation and estradiol binding of estrogen receptor in hormone-dependent and hormone-independent GR mouse mammary tumors.

The effect of phosphorylation on the hormone-binding capacity of the estrogen receptor (ER) was investigated in hormone-dependent (HD) and hormone-independent (HI) mammary carcinomas of GR mice. Tumor cytosols were incubated with ATP under conditions previously used to study the tyrosine kinase which confers hormone binding to phosphatase-treated or in vitro-synthesized ER. The ATP-dependent increases in hormone-binding capacity of 8 out of 20 HI tumors ranged from values of 23 to 124 fmol/mg cytosol protein. The enhancement by ATP of hormone binding to ER was significantly less marked in HD and HR tumors than in HI tumors. In only 3 out of 13 HD and HR tumors was an increase ranging from 15 to 20 fmol/mg protein detected. Analysis by Scatchard plot of estradiol binding to ER showed that cytosol incubation of HI tumors with ATP markedly increased the hormone binding without any change in affinity. The data suggest that ER of HI tumors is less phosphorylated in vivo than the ER of HD/HR tumors, so that the receptor of HI tumors is more susceptible to gamma-32P-ATP phosphorylation and ATP-induced hormone binding in vitro. Western blot of ER with antiphosphotyrosine antibody showed that, in HI tumors, the large ATP-induced increase in hormone binding to ER was associated with phosphorylation on tyrosine of the receptor itself. Our findings indicate that the process of activation-inactivation of binding through tyrosine-phosphorylation/phosphotyrosine-dephosphorylation of ER observed in estrogen target tissues is altered in some HI mammary tumors.

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.

Lipid Metabolism and Enzyme Activities in Hormone-Dependent and Hormone-Independent Mammary Adenocarcinoma in GR Mice&lt;xref ref-type="fn" rid="FN1"&gt;2&lt;/xref&gt;

Lipid metabolism in hormone-dependent (HD) GR mouse mammary tumors was compared to that in hormone-independent (HI) tumors and normal mammary tissues. HD tumors, like normal mammary tissue but unlike HI tumors, synthesized medium-chain-length fatty acids (MCFA). However, when treated with hormones (estrone and progesterone), the HI tumors were induced to produce MCFA. The activity of thioesterase II correlated positively with the synthesis of MCFA and was influenced by the hormones administered. The activities of NADP+-linked malate dehydrogenase, citrate lyase, acetyl-CoA carboxylase, and fatty acid synthetase, although lower in tumors than in normal glands, were not different in HD as compared to HI tumors. Whereas the predominating lipids synthesized in normal glands were triglycerides, phospholipids comprised about half of the lipid synthesized in the tumors, with no difference between HD and HI tumors. The conversion of D-[U-14C]glucose to 14CO2 was higher in HD tumors than in HI tumors but increased in HI tumors treated with hormones in vivo. By a comparison of the 14CO2 produced from D-[1-14C]glucose and from D-[6-14C]glucose in the presence and absence of an electron acceptor (methylene blue), it was demonstrated that regeneration of NADP+ from NADPH was a rate-limiting step for the pentose phosphate pathway in the tumors. Hence, while differences in the lipid metabolism can be demonstrated between HD and HI GR mouse mammary tumors, some of the changes are due to the hormone treatment rather than to a specific alteration in the tumor itself.

Histone phosphorylation in explants of mouse mammary glands and tumors

Rates of histone phosphorylation were measured in explants of mammary glands from mouse strains with high and low tumor incidence. Explants of hormone dependent and independent mouse mammary tumors were also investigated. All mouse strains studied showed predominant phosphorylation of H2A histone at serine and threonine residues. No differences in rates of H2A phosphorylation in glands were found between strains having different mammary tumor susceptibility. Hormone-dependent GR mouse mammary tumors also showed high H2A phosphorylation, but in some tumors also H1 and H3 were phosphorylated. Hormone-dependent GR tumors had 2-5 times higher histone phosphorylation at serine and threonine than hormone-independent tumors.

Chemotherapy and hormonal therapy of mouse mammary tumors

Single Cisplatin (Platinol®) injections i.p. at 10 mg/kg dose level (LD10) caused a marked growth inhibition of both hormone dependent and independent GR mouse mammary tumors (reduction in tumor volumes of 86 and 85%, respectively). However this treatment caused severe toxic side effects including loss of mouse body weight and deaths of some of the animals. When Cisplatin (pure) was administered s.c. in a single cholesterol pellet, no such toxic effects were observed even when Cisplatin levels up to 30 mg/kg were applied. The latter dose caused 29% reduction in tumor volume of hormone independent tumors. However this inhibitory effect was temporary, since regrowth of the hormone independent tumors started again 8 days after application of the single Cisplatin pellet. These results show that continuous Cisplatin administration in pellets s.c. is less toxic than injection i.p., but that the inhibition of mammary tumor growth is also less marked.

Translocations involving chromosome 1 in hormone-independent GR mouse mammary tumors

Large marker chromosomes were found in 2 hormone-independent (HI) GR mouse mammary tumors, i.e. a tumor (TSl 75) that was already HI at the first transplantation, and a tumor (TSl 66) that became HI at the 19th passage. These marker chromosomes appear to be translocations to distal and proximal ends of chromosome 1, respectively. The translocation in tumor TSl 66 may be t(15:1).

The ability to concentrate iodide as a marker of hormone dependence in GR mouse mammary tumors.

We have previously shown that hormone-dependent (HD) but not hormone-independent (HI) mammary tumors of GR mice concentrate iodide. Since hormone dependence is lost during serial transplantation of the tumors, we have investigated whether the ability to concentrate iodide is lost simultaneously. All tumors progressing to hormone independence lost the ability to concentrate iodide at the time of transition from the HD to the HI state. We believe, therefore, that the ability to concentrate iodide is a marker of hormone dependence in GR mouse mammary tumors.

348 Chromosomal aberrations in GR mouse mammary tumors during their progression from hormone dependence to independence

348 Chromosomal aberrations in GR mouse mammary tumors during their progression from hormone dependence to independence

387 Changes in nuclear protein and hormonal dependency in GR mouse mammary tumors

387 Changes in nuclear protein and hormonal dependency in GR mouse mammary tumors

Autoradiographic evidence of a loss of iodination within hormone-dependent GR mouse mammary tumors as they progress to independence.

The purpose of this study was to determine by use of light- and electron-microscope autoradiography whether or not iodination occurred in mammary tumors of female GR mice. Of the sixty tumors studied it was found that pregnancy-dependent and hormone-induced tumors possessed iodinating ability. Although mammary glands from nonpregnant GR mice lacked the ability to iodinate, by the 16th day of pregnancy in response to hormonal stimulation the glands readily iodinated casein, and some epithelial cells contained ultrastructural cytochemical evidence of mammary peroxidase. Preneoplastic mammary gland lesions known as hyperplastic alveolar nodules were also able to iodinate, as were plaques, the disc-shaped lesions which give rise to the hormone-responsive mammary tumors in this strain. Plaques also contained epithelial cells with mammary peroxidase activity. When hormone-induced mammary tumors were transplanted into syngeneic mice they retained the ability to iodinate for several generations. However, as the tumors progressed to hormone independence, the ability to iodinate was gradually lost. Hormone-independent mammary tumors from GR mice lacked both iodinating ability and cytochemical evidence of mammary peroxidase. These findings suggest that iodination depends upon hormone-responsive cells within the mammary tumors and that as these cells become hormone unresponsive, the ability to iodinate is lost.

Cyclic Biological Expression in Mouse Mammary Tumors

Biological characteristics were assessed in GR mouse mammary tumors during 22 serial transplantations. Although unidirectional progression from hormone dependency to independency was observed, other biological markers such as progesterone receptors, polyploid frequency and thymidine kinase activity demonstrated cyclic phenomena every fourth to sixth transplant generation, suggesting the continued presence of regulatory mechanisms among various cells subpopulations.

The region of mouse mammary tumor virus DNA containing the long terminal repeat includes a long coding sequence and signals for hormonally regulated transcription.

Starting from a biologically active recombinant DNA clone of exogenous unintegrated GR mouse mammary tumor virus, we have generated three subclones of PstI fragments of 1.45, 1.1, and 2.0 kb in the plasmid vector PBR322. The nucleotide sequence has been determined for the clone of 1.45 kb which includes almost the complete region of the long terminal repeat (LTR) plus an adjacent stretch of unique sequence DNA. A short region of the 2.0 kb clone, containing the beginning of the LTR, has also been sequenced. Starting with the A of an initiation codon outside the LTR, we detected an open reading frame of 960 nucleotides, potentially coding for a protein of 320 amino acids (36K). Two hundred nucleotides downstream from the termination codon, and approximately 25 nucleotides upstream from the presumptive initiation site of viral RNA synthesis, we found a promoter-like sequence. The sequence AGTAAA was detected approximately 15-20 nucleotides upstream from the 3' end of virion RNA and probably serves as a polyadenylation signal. The 1.45 kb PstI fragment has been transfected into Ltk- cells together with a plasmid containing the thymidine kinase gene of herpes simplex virus. The virus-specific RNA synthesis detected in a Tk+ cell clone was strongly stimulated by the addition of dexamethasone.

Estrogen responsive creatine kinase in normal and neoplastic cells

Estrogen-responsive creatine kinase (uterine estrogen-induced protein, CK-BB) activity was compared during ontogeny of the rat uterus, in the human menstrual cycle and in mouse and human mammary tumors. Between days 2 and 26 of post-natal development of rat uterus, the specific activity of creatine kinase increases 4.5-fold. The glycolytic enzymes, phosphoglycerate kinase and phosphoglycerate mutase, show no increase and pyruvate kinase activity increases 1.5-fold, during this period. Only creatine kinase BB activity was increased by estrogen administration. In human endometrium, the specific activity of creatine kinase increases in the late-secretory stage of the menstrual cycle. During the progression from hormonal dependence to independence shown by the GR mouse mammary tumor in the course of successive transplantations, the total activity of creatine kinase increases. The increase in the MM (muscle type, non-estrogcn responsive) isozyme activity of creatine kinase exceeds the increase in creatine kinase BB activity. Normal human breast and breast tumors display both creatine kinase isozymes. Preliminary evidence for in vitro estrogen responsiveness of creatine kinase, in human breast explants, raises the possibility that creatine kinase BB may be a suitable marker for assessing the hormonal dependence of human tumors.

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.

Outgrowth of grafts containing different ratios of hormone-dependent and independent mouse mammary tumor cells.

The growth properties of grafts composed of different mixtures of hormone-dependent and independent GR mouse mammary tumor cells were investigated. Grafts containing only hormone-dependent mammary tumor cells yielded tumors in 4--6 weeks in estrone plus progesterone treated castrated mice, but not in castrated mice that were not treated with hormones. Grafts containing only autonomous mammary tumor cells yielded tumors in about 2 weeks in both hormone-treated and untreated castrated mice. Grafts containing mixtures of hormone-dependent and autonomous cells yielded tumors in 2 weeks both in hormone-treated and untreated castrates, even when the grafts contained predominantly hormone-dependent cells (up to 90%). These results indicate that the growth behavior of heterogeneous mammary tumor grafts that contain more than 10% autonomous cells, is essentially determined by these cells. Growth of the autonomous cells is not markedly affected by the presence of hormone-dependent cells in the grafts.

A unique glycoprotein containing GR-mouse mammary tumor virus peptides and additional peptides unrelated to viral structural proteins

A glycoprotein of molecular weight 130,000 (gP130) has been precipitated from the cytoplasm of GR-strain mouse mammary tumor (GR-MMT) cells by a rabbit antiserum (anti-MMTV) to GR-strain mouse mammary tumor virus (GR-MMTV). This protein was not precipitated by antisera specific for detergent-disrupted C3H-strain MMTV (C3H-MMTV); C3H-MMTV glycoproteins; C3H-MMTV nonglycosylated proteins; GR-MMTV p25 or p12; RIII strain (milk) MMTV proteins; or Rauscher murine leukemia virus (R-MuLV) proteins; nor was it precipitated by normal rabbit serum. Two-dimensional thin layer analysis of 35S-methionine-containing tryptic peptides revealed that five of nine gp33 peptides and one of seven gp55 peptides are shared by gP130 and gPr76 env. The envelope protein precursor, gPr76 env, contains all of the gp33 peptides and six of seven gp55 peptides. One peptide in gPr76 env, possibly a gp55-gp33 junction peptide, is also apparently present in gP130. Six of ten p25 peptides and four more gag-related peptides are shared by Pr78 gag and gP130. Protein gP130 also contains several tryptic peptides not found in gPr76 env or in the core protein precursors Pr78 gag, Pr110 gag or Pr180 gag-pol. Radioimmunoprecipitation experiments showed that gP130 could be precipitated from extracts of GR-MMTV cells with anti-MMTV serum even after antibodies to the known MMTV structural proteins had been removed from the serum by absorption. Both gP130 and a second protein, p30, were found in immunoprecipitates of detergent-disrupted isotopically labeled GR-MMTV treated with the absorbed anti-MMTV serum. These results suggest that antibodies to gP130 in the anti-MMTV serum are capable of recognizing those protein sequences unique to gP130; that is, those protein sequences which are not related to viral structural proteins. In light of these data and data published previously, gP130 is apparently a polyprotein containing juxtaposed components translated from the 5′ and 3′ end of the MMTV genome and protein components not previously identified as virus-specific.

Detection and characterization of mouse mammary tumor virus cell surface antigens: estimation of antigen abundance by protein A assay.

Antisera against the following mouse mammary tumor virus (MMTV) structural proteins were used to detect MMTV cell surface antigens: (i) the 27,000-dalton nucleoid protein, p27; (ii) the 36,000-dalton envelope glycoprotein, gp36; and (iii) the 52,000-dalton exterior envelope glycoprotein, gp52. We report here the development of an adherent-cell isotopic staphylococcal protein A (SPA) test (ISPAT) for MMTV structural proteins which allows for the detection of an MMTV membrane-associated antigen as well as an estimate of its relative abundance on the cell surface. This test demonstrated that the gp52 was the predominant MMTV cell surface antigen detected on both C3H and GR mouse mammary tumor cells. In a comparative study with anti-gp52 and anti-gp36 sera, SPA-specific binding with anti-gp36 serum was found to be only 5 to 6% of that obtained for the external virion glycoprotein, gp52. Both direct and indirect ISPAT indicated the presence of a low but detectable number of gp36 determinants on GR-MMTV cells; however, these gp36 determinants, unlike gp52 determinants, appeared to be exposed by the fixation procedure used. Only 0.9 to 1.1% of the gp52-specific binding was detected when anti-gp36 serum was allowed to react with viable cells. The binding of [125I]SPA achieved with anti-p27 serum was even less than that detected with gp36-directed reagents, indicating that p27 is not a cell surface antigen. The use of fluoresceinated SPA further demonstrated that p27 and gp36 reactivity was only associated with a small number of cells in each of the mammary cultures tested. When N-[4-(5-nitro-2-furyl)-2-thiazoly]-formamide-induced C3H bladder tumor cells were subjected to a gp52-directed ISPAT, the failure to detect gp52-specific binding demonstrated the specificity of this assay for MMTV gp52-expressing cells. In addition to detecting and characterizing MMTV cell surface antigens, the newly developed adherent cell assay could measure changes in the abundance of cell surface gp52. When dexamethasone-treated and untreated GR cells were compared, measurements of gp52-specific SPA binding indicated that dexamethasone stimulation leads to a 12.2-fold increase in the amount of cell surface gp52 detected.