Characterization of rare mammary tumours appearing on the neck of RIII/Sa mice infected with mouse mammary tumour virus.

RIII/Sa and C3H mice harbour milk-borne mouse mammary tumour virus (MMTV) and develop mammary tumours at a high incidence. These mammary tumours usually arise ventrally and/or on the sides of the animals. In the present study, some mice of both strains were observed to have tumours in the dorsal neck area. Histological analysis of the tumours indicated their similarity to mammary tumours induced by MMTV oncogenesis. The neck tumours were found by thin-section electron microscopy to contain both type A and type B particles that are hallmarks of MMTV infection. In addition, the neck tumour DNA possessed insertion mutations of Wnt-1 and Fgf-3 proto-oncogenes, the activation of which play important roles in the development of mouse mammary tumours. These neck tumours appear to be mammary tumours that arise in the context of in-situ mammary tissue, similar to rare 'ectopic' human breast cancers that arise in the axillary region and other sites remote from the breast.

Overexpression of the p53 gene product in canine mammary tumors.

p53, a tumor suppressor gene, is a target of genetic alternations in many human and animal cancers. Compared to normal tissues, cancer tissues overexpress mutant p53 protein thus allowing their detection by a number of immunochemical procedures. To what extent the expression of mutant p53 correlates with dog mammary tumorigenesis has not been fully studied. In the present study, 20 spontaneously arising canine mammary tumors were examined for overexpression of mutant p53. Two different monoclonal antibodies, BP53-12 and PAb122, which recognize different epitopes of the p53 product, were used. The canine tumors in the present study exhibited five different histological types: i) osteosarcoma (n=7); ii) carcinosarcoma (n=4); iii) solid carcinoma (n=5); iv) complex carcinoma (n=3); and v) tubulopapillar carcinoma (n=1). The positive ratios against BP53-12 and PAb122 antibodies were 50% (10/20) and 60% (12/20) respectively. Among these positive samples, 35% (7/20) reacted to both antibodies. Finally, 15 out of 20 tumors showed positivity against one of the monoclonal antibodies. Mostly, as in human mammary tumor cells, BP53-12 staining was observed in the nuclei of tumor cells. PAb122 staining, however, was confined to cytoplasm of osteosarcoma or carcinosarcoma cells. To confirm the location of the staining, immunoelectron microscopy was done. The results showed that the cytoplasm of cartilage cells in the sarcomas had positive staining. These results indicate that anti-p53 antibodies BP53-12 and PAb122, generated against human p53 are cross reacting with the same molecule in canine cells and that the role of p53 in tumorigenesis is not only confined to tumors in human. Our finding suggests that a combination of p53 monoclonal antibodies should be used to screen, not only canine mammary tumors but also human mammary tumors, to obtain a better tumor prognosis.

Cloning of an infectious milk-borne mouse mammary tumor virus (MMTV) DNA from a mammary tumor that developed in an endogenous MMTV-free wild mouse.

Molecular characterization of infectious mouse mammary tumor viruses (MMTVs) has been hampered due to the problem of cloning a full-length exogenous virus into a plasmid. The present report describes our strategy for obtaining a full-length clone of an exogenous MMTV from a mouse mammary tumor that arose spontaneously in a wild Chinese mouse free of endogenous MMTV and shows that the cloned virus (JYG-MMTV) is expressed in rat RBA cells. Four-week-old C58/J x CBA/CaJ female mice, free of both endogenous and exogenous MMTVs, were injected with virus-secreting RBA cells. The progeny of these mice were bred, and their offspring were tested for the presence of MMTV. These third-generation mice were found to actively produce MMTV that was shed in their milk and transmitted to their offspring. The virus was detected not only in the mammary glands of these young mice, but also in their spleens and bone marrow. These results suggest that our plasmid-cloned exogenous JYG-MMTV is infectious. This virus can now be used effectively in manipulating the various genes of JYG-MMTV and other MMTV strains to understand their structure/function relationships.

Antisense downregulation of a mouse mammary tumor virus activated protooncogene in mouse mammary tumor cells reverses the malignant phenotype.

Activation of the protooncogene Wnt-1 by insertion of the mouse mammary tumor virus (MMTV) is known to cause mammary tumors in mice. Wnt-1 expression in mammary glands has been postulated to confer direct local growth stimulation of mammary epithelial cells leading to their acquisition of a preneoplastic state. Wnt-1 expression also induces morphological alterations in cultured normal mammary cells. However, it has not been determined whether or not transformed mammary cells require continuous Wnt-1 expression for their ability to form tumors in vivo. To address this question, we constructed antisense and sense Wnt-1 expression vectors containing a synthetic promoter composed of five high-affinity glucocorticoid response elements (GRE5). This promoter is at least 50-fold more inducible by dexamethasone than the promoter contained in the long terminal repeats of MMTV. The vectors were introduced into a mouse mammary tumor cell line (R/Sa-MT) that expresses high levels of endogenous Wnt-1 mRNA and forms rapidly growing tumors when transplanted into syngeneic hosts. Of the 12 stably transfected cell lines established (9 with antisense and 3 with sense constructs), 2 antisense cell lines (R/Sa-MT/antisense) and 1 sense cell line (R/Sa-MT/sense) were examined for inducibility by dexamethasone of antisense and sense Wnt-1 RNAs, changes in endogenous Wnt-1 RNA expression, and changes in cell morphology. The growth patterns of the cells in vitro and in vivo were also examined. Our results show that (1) the levels of the expression of endogenous Wnt-1 mRNA and protein were reduced significantly (>80%) in those cells (R/Sa-MT/antisense) that expressed antisense Wnt-1 RNA at high levels following exposure to dexamethasone, compared to the R/Sa-MT/sense and R/Sa-MT control cells and (2) transplantation of the R/Sa-MT/antisense cells produced smaller tumors ( approximately 0.2 cm in 16 weeks) compared to the tumors ( approximately 2.0 cm in 8 weeks) that were produced by the R/Sa-MT/sense and R/Sa-MT cells. We therefore suggest that Wnt-1 expression is required not only for the transformation of normal mammary cells into tumor cells, but also for the maintenance of their tumorigenicity.

Clonal variations among multiple primary mammary tumors and within a tumor of individual mice: insertion mutations of int oncogenes.

Laboratory mice infected with the mouse mammary tumor virus (MMTV) often develop multiple mammary tumors. However, no comprehensive studies have been done addressing the question of whether or not different primary tumors of individual mice are related ontogenetically to each other. Further, it is not known to what extent individual tumors vary in their cellular composition. We, therefore, examined intertumor and intratumor patterns of the rearrangements in int-1 (Wnt-1), int-2 (Fgf-3), and int-3 protooncogenes, since mutations in ints caused by MMTV result in the development of mammary tumors in mice and thus provide the most suitable genetic markers for the tumor cells. Our results show that, irrespective of the genetic background of the mice and/or the strain of MMTV, the pattern of MMTV integration in the different tumors of individual mice varies as widely as is found with the tumors of different mice. Of the 79 tumors obtained from 25 mice of different genetic backgrounds, 31 showed insertional mutations in int-1 (39%). By contrast only 9 of the 65 tumors tested had mutations in int-2 (14%). None of the tumors showed mutations in int-3. Interestingly, tumors from individual mice also showed variations in their pattern of int gene mutation, indicating that multiple tumors that develop in a mouse bear no ontogenetical link. The analysis of the pattern of intratumor MMTV integration, int mutation, and int expression revealed that several int mutational events as well as variations in int expression may occur in the same tumor. The diversity of int activation within individual tumors may suggest that the initiation and progression of most mammary tumors, if not all, occur through the concerted action of different mutational events in the same cell or through interactions of different cell populations, each of which acquired different int mutations.

Characterization and chromosomal localization of the human homologue of a rat AMP-activated protein kinase-encoding gene: a major regulator of lipid metabolism in mammals.

AMP-activated protein kinase (AMPK) phosphorylates and inactivates acetyl-CoA carboxylase and beta-hydroxy beta-methylglutaryl-coenzyme A (HMG-CoA) reductase which are the major enzymes involved in fatty acid and lipid biosyntheses. The AMPK gene from rat (rAMPK) has recently been cloned [Carling et al., J. Biol. Chem. 269 (1994) 11442-11448]. In order to study the structure and function of the human AMPK gene (hAMPK), we have cloned the gene, and report in this communication its nucleotide (nt) sequence, tissue distribution and chromosomal location. Our results show that the ORF of hAMPK encodes 552 amino acids (aa) (62.250 kDa) and is highly conserved with rAMPK with identities of 97.3 and 90% at the aa and nt levels, respectively. The hAMPK gene bears homology to a yeast protein kinase-encoding gene (snf1) that regulates carbohydrate metabolism, and also with three other genes encoding SNF1-like kinases from different plant species, namely Arabidopsis thaliana, Hordeum vulgare and Secale cereale. As determined by fluorescent in situ hybridization of a human metaphase chromosome spread, hAMPK maps to chromosome 1p31. The size of the hAMPK transcript is 8.5 kb and the transcription start point (tsp) is located approx. 46 bp upstream from the ATG codon. While 10-15% of AMPK is alternatively spliced in most tissues of the rat, our RT-PCR analyses of the hAMPK mRNA did not reveal the presence of any alternatively spliced form of the gene in human tissues. An interesting aspect of AMPK is that its expression, unlike in rat liver, could not be detected in human liver, and thus the purported role of the gene in controlling fatty-acid synthesis in the human liver remains to be determined.

Cloning in a plasmid of an MMTV from a wild Chinese mouse: sequencing of the viral LTR.

Plasmid subcloning by conventional techniques of full length exogenous mouse mammary viruses (MMTV) has not been realized because of the involvement of host-mediated structural changes in the viral gag gene. To circumvent this problem, an alternative subcloning method, excision of phagemid (pBluescript SK) from lambda ZAP II, was successfully used to subclone a novel exogenous MMTV (JYG-MMTV) provirus fragment containing an intact gag gene. Sequence analysis revealed that the LTR of this virus is significantly different from the LTR of C3H-MMTV in the U3 region.

Insertional mutation of int protooncogenes in the mammary tumors of a new strain of mice derived from the wild in China: normal- and tumor-tissue-specific expression of int-3 transcripts.

A new mouse strain, Mus musculus Jyg, has been isolated from the wild in China. After several generations of inbreeding, Jyg mice have been found to develop mammary adenocarcinomas at a high incidence (70-80%). In order to understand the mechanism by which mammary tumors are induced in these mice, we analyzed 23 available mammary tumors and liver tissues with regard to mouse mammary tumor virus (MMTV) proviral integrations and the pattern of int oncogene (Wnt-1, int-2/Fgf-3, and int-3) rearrangements and expression. We found that (1) Jyg mice do not carry endogenous MMTV; (2) all tumors showed multiple MMTV proviral integrations and expressed high levels of MMTV; (3) Jyg MMTV is distinguishable from other MMTV strains; (4) a high percentage of the tumors (70%) had insertional mutations in int loci (Wnt-1, 26%; int-2, 13%; and int3, 43%); and (5) unlike Wnt-1 and int-2, a 5.9-kb int-3-related transcript is expressed in developing mouse embryos of all stages and adult mouse tissues including mammary tumors, whereas a 2.4- to 3.6-kb transcript is expressed only in Jyg mammary tumors with int-3 mutations. Taken together, this newly developed mouse strain and the milk-borne MMTV that it carries constitute a novel system for studies of the host and viral specificity of insertional mutagenesis of multiple int protooncogenes by MMTV and the role of these genes in the pathogenesis of mouse mammary carcinomas and tumor cell heterogeneity.

Dietary regulation of mammary tumorigenesis in RIII/Sa mice: investigation of a possible mechanism.

The effects of caloric restriction on the incidence of mammary tumor development, the levels of the expression of mouse mammary tumor virus (MMTV)- and prolactin-RNA, as well as the levels of serum prolactin, were investigated in virgin RIII/Sa mice, a strain known to display a high incidence of spontaneous mammary tumor development. Of the 54 mice fed a low-calorie (LC; 10 kcal/day) diet containing low fat (LF; 5% corn oil) for a period of 72 weeks, only seven mice were found to develop mammary tumors, an incidence of 13%. By contrast, the cumulative tumor incidence in a similar sized group of mice, fed a high-calorie (HC; 16 kcal/day) low fat-containing diet was 73%. Estimation of the relative levels of MMTV-RNA, as determined by Northern and slot blot hybridizations, in the mammary glands of mice fed LCLF and HCLF diets for 8, 10, 16, 28, and 36 weeks revealed that the LCLF diet-fed mice expressed 4-15-fold less RNA than the HCLF diet-fed mice. Interestingly, however, the LCLF diet did not appear to exert any effect on the expression of prolactin RNA even though it reduced the levels of serum prolactin. We suggest that in RII/Sa mice the modulation of MMTV-induced mammary tumors by dietary calorie is linked to the secretion of serum prolactin which, in turn, affects the replication of MMTV required for mammary cell transformation.

Distribution of mouse mammary tumor virus in Asian wild mice.

Several groups of wild mice (Mus musculus) were captured from eight different locations in Asia and bred for several generations in a facility free of any laboratory strains of mice carrying mouse mammary tumor virus (MMTV). The distribution of endogenous MMTV proviral sequences in the liver tissues of these mice was investigated by using Southern blot hybridizations. Four categories of mice were identified. Mice originating from Bogor, Indonesia (Cas-Bgr); He-mei, Taiwan (Cas-Hmi/1); and Malaysia (Cas-Mal) were found to carry an endogenous MMTV provirus consisting of the env, gag-pol, and long terminal repeat sequences. Mice captured from Kojuri, Republic of Korea (Sub-Kjr); Nagoya, Japan (Mol-nag); and three Chinese provinces, Shanghai (Sub-Shh), Beijing (Sub-Bjn), and Jiayuguang (Sub-Jyg/1), appeared to carry defective proviruses. Some mice originating from He-mei (Cas-Hmi/2) and Jiayuguang (Sub-Jyg/2) were found to be completely free of endogenous MMTV. Interestingly, however, the Sub-Jyg/2 mice, after several generations of inbreeding, were found, unlike all of the other subspecies that we examined in the present study, to develop mammary tumors at a high incidence (80 to 90%) with a short period of latency. Electron microscopic examination of the mammary glands and mammary tumors of these mice revealed the presence of numerous intracytoplasmic A, immature, budding, and mature B particles. Furthermore, the mammary tumors were found to contain MMTV proviral sequences. It seems, therefore, that Sub-Jyg/2 mice carry an exogenous MMTV which contributes to their developing mammary tumors.

Differential induction of c-jun expression by PGF2-alpha in rat ovary, uterus and adrenal.

The influence of PGF2 alpha on c-jun gene expression in ovary, uterus and adrenal was examined. Three and seven day postovulatory PMSG primed immature rats received 500 ug PGF2 alpha by two subcutaneous injections 8 hours apart. Control rats received saline. Animals were sacrificed 30 minutes after the second injection of PGF2 alpha. Tissues were obtained and frozen in liquid nitrogen. RNA extracted from ovary, uterus and adrenal was analyzed by Northern and slot blot. c-jun was expressed differentially in these organs. An increase in c-jun expression by PGF2 alpha treatment occurred in the ovary but not in the adrenal and uterus. The effect of PGF2 alpha on c-jun was stronger in older compared to younger corpora lutea. These results indicate differential regulation of c-jun by PGF2 alpha in steroidogenic and steroid responsive tissues and that c-jun might be linked to the mode of action of PGF2 alpha in luteolysis.

Polyclonal and monoclonal antibodies monospecific to MMTV LTR orf protein produced in E. coli.

Monoclonal and polyclonal antibodies specific to an open reading frame of the mouse mammary tumor virus long terminal repeat were generated using an open reading frame-beta-galactosidase fusion protein produced in E. coli. Both antibodies reacted with the open reading frame-beta-galactosidase fusion protein but not with beta-galactosidase alone using an immunoblotting technique. It is concluded that these antibodies were specific for the protein encoded by the open reading frame of the mouse mammary tumor virus long terminal repeat.

Mixed inocula of mouse mammary tumour cell subpopulations result in changes of organ-specific metastasis.

Tumour and metastatic phenotypes, the pattern of mouse mammary tumour virus (MMTV) integration and expression, and the expression of a metastasis associated gene, nm23, were examined in three mammary tumour cell subpopulations, 66, 168 and 4526. Tumour growth, host survival, metastatic aggressiveness, and the distribution of different cell types in metastasis resulting from mixed cell inocula were also analysed. The results of these studies indicated that the cell lines were distinguishable from each other both phenotypically and genotypically. However, a rearrangement of the mammary tumour specific protooncogene, int-1, caused by MMTV was found to be a unique characteristic of the cell line 4526. Therefore, int-1 was used as a stable marker to examine the genotype of the metastatic colonies that developed in mice bearing tumours of mixed cell inocula. Highly metastatic 4526 cells influenced the metastatic range of poorly metastatic 66 cells. Line 66 cells that normally colonize only to lungs were also found to colonize liver when inoculated together with the liver-metastasizing 4526 cells. This acquired metastatic phenotype of 66 cells was transient. On the contrary, mixed cell inocula of 4526 and non-metastatic 168 cells did not produce any colony of 168 cells. The metastatic aggressiveness of 4526 cells was inhibited by both 66 and 168 cells. Furthermore, the metastatic behaviour of mixed inocula differed depending on the relative abundance of the component populations in the mixtures. These findings suggest that interaction between cells of different metastatic phenotypes may result in changes of their metastatic behaviour.

Colonization characteristics of a murine mammary tumor cell line that metastasizes frequently to the heart.

Metastasis by mouse mammary tumor cells is usually confined to lung. This paper describes the metastatic behavior of an established mouse mammary tumor cell line, 4526, that in addition to lung and liver metastasis, shows a high rate of heart metastases. The tumor cells were inoculated into the fourth mammary fat pad of syngeneic mice and their pattern of distant colonization was analysed qualitatively as well as quantitatively. We found that the cell line produced 100, 70 and 40% metastases to the lung, liver and heart, respectively. While the lung metastases appeared primarily as nodular masses, the liver metastases occurred both as nodular and diffuse masses. In addition, we observed that the metastatic load of each of the different lung lobes of individual mice was proportional directly to its relative size, and there seemed to be an inverse relationship between the occurrence of lung and liver metastases in individual mice. As compared to lung and liver metastases, heart metastases were found to be localized internally, usually in the cavity and wall of the ventricle. Furthermore, hearts with metastases revealed destruction of cardiac tissue and blockage of the cavity space. Our results show that 4526 cells are phenotypically stable, since the metastatic behavior of several clonal derivatives of the cell line obtained from lung, liver and heart colonies were found to be identical to that of the parental cell line. Thus this cell line, because of its unparalleled metastatic characteristics, offers a model for investigations into the biology of mammary tumor cell metastasis, especially heart metastasis.

The interaction of a c-Jun/Fos related protein factor with the U3 sequences of the mouse mammary tumor virus LTR.

Using polyacrylamide gel mobility shift assay we have detected a nuclear factor (NF-S) in a mouse mammary tumor cell line (GR) that binds to an upstream sequence domain (-766 to -737) near the 5'-end of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). Antibodies to the products of the Jun and Fos oncogenes interfered with the binding potential of this factor, indicating that the factor shares antigenic determinants with c-Jun/AP-1. In vitro translated c-Jun and c-Fos were also found to bind to the NF-S binding domain consisting of the sequence TGA(A/G)TCA that are known to be recognized by c-Jun/AP-1. Our results raise the possibility that the MMTV-LTR sequence element-766 to -737 by interacting with a Jun/Fos related protein play a role in MMTV transcription and/or the activation of int protooncogenes that are associated with murine mammary tumorigenesis.

Genetic diversity of spontaneously developed primary and metastatic mammary tumor cells in mice.

Metastatic cells are often considered to be clonal derivatives of one of the primary tumor cell subpopulations. To determine if the cells of spontaneously developed lung nodules in a mammary tumor-bearing mouse represent the major or minor population of the cells in the primary tumor, comparisons were made of the pattern of their mouse mammary tumor virus (MMTV) proviral integrations, and their insertional mutations of the mouse mammary tumor proto-oncogenes, int-1 and int-2. Of the 78 tumor-bearing C3H/He mice sacrificed, seven mice showed metastatic lung nodules, but only four mice had nodule tissues adequate for the present analysis. Examination of the primary tumors and the corresponding lung nodules of these four mice revealed that the number of newly integrated MMTV proviruses and their sites of integration in the genomic DNAs of primary tumors and tumor nodules were variable, and that the int-1 gene was disrupted in three of the primary tumors but not in any of the metastasized tumor tissue. These results support the notion that, at least in some mice, the majority of cells constituting the primary mammary tumors and the corresponding metastases are of different genotypes and raise the possibility that the activation of different int-proto-oncogenes may be involved in the genesis of different cell subpopulations including metastatic cells in the same mouse.

Expression of the oncogenes mil and ras abolishes the in vivo differentiation of mammary epithelial cells.

Three carcinoma-associated oncogenes, two of which have been strongly implicated in human mammary tumorigenesis, have been introduced into a novel mouse mammary epithelial cell line, EF43, that retains many differentiated functions. The effect of oncogene expression upon classical transformation parameters as well as parameters specific for mammary epithelial cells such as growth in three-dimensional collagen matrices and the ability to repopulate the cleared mammary fat pad and to form alveolar structures in vivo has been investigated. Expression of v-myc in EF43 cells results in no obvious phenotypic changes, and does not confer tumorigenic potential upon the cells. Expression of v-Ha-ras confers upon EF43 cells the ability to grow rapidly, grow in an anchorage-independent manner, results in tumor formation in nude and syngeneic animals, abolishes their ability to repopulate the mammary gland and, instead, results in rapid induction of anaplastic tumors. The v-mil oncogene, an avian homolog of the mouse v-mht and human c-raf oncogenes, previously thought to be non-transforming in the absence of a co-operating oncogene, transforms EF43 cells, allowing them to grow in an anchorage-independent manner, form tumors in nude mice and abolishes their ability to repopulate the cleared mammary fat pad. In contrast to v-ras, however, the tumors arising from v-mil expression have a differentiated morphology, typical of adenocarcinomas. Thus, different oncogenes show varying degrees of inhibition of the differentiation of mammary epithelial cells in vivo.

Structural alterations in the long terminal repeat of an acquired mouse mammary tumor virus provirus in a T-cell leukemia of DBA/2 mice.

ML, a transplantable T-cell leukemia of DBA/2 mice, expresses the gag and env gene products of the murine mammary tumor virus (MuMTV). Analysis of the genomic DNA of ML cells using the restriction enzyme HindIII and hybridization with MuMTV-specific probes revealed that the ML cells contained two or more newly integrated MuMTV proviruses (ML-MuMTV). Further analysis of these proviruses with a combination of Mspl and Pstl enzymes showed that the long terminal repeat (LTR) (ML-MuMTV LTR) of the ML-MuMTV provirus(es) was structurally different from the LTRs of both exogenous and endogenous MuMTV proviruses of DBA/2 mice. In order to characterize the nature of the structural alterations in the ML-MuMTV LTR, we cloned a 4.0-kb HindIII fragment containing the 3' half of an acquired provirus. Sequence analysis of the ML-MuMTV LTR of this acquired provirus revealed a deletion of a 387-bp segment that maps between the 5' nucleotide 616 and the 3' nucleotide 1003 of the normal MuMTV LTR and duplication of a 102-bp fragment that mapped between 514 and 616. In addition to two point mutations in the direct repeat, the proviral ML-MuMTV LTR has also acquired 9- and 7-bp segments at the 5' and 3' sites of the duplicated 102-bp segment, respectively. Since direct repeats in the U3 regions of a number of LTRs have been found to be associated with enhancer function, we examined the enhancer function of the U3 region sequences of the ML-MuMTV LTR using enhancer-dependent transient expression assay of chloramphenicol acetyltransferase (CAT) gene in NIH 3T3 cells. Our studies have shown that the U3 region sequences of the rearranged ML-MuMTV LTR have the ability to enhance the expression of the CAT gene 12- to 15-fold more than the U3 region sequences from the normal MuMTV LTR. The presence of a direct repeat in the ML-MuMTV LTR and its ability to enhance the transcription of adjacent genes is analogous to the LTRs of certain murine leukemia viruses.

The effects of 2-deoxyglucose and tunicamycin on the biosynthesis of the murine mammary tumor virus proteins, and on the assembly and release of the virus.

The role of glycosylation in the biosynthesis, processing, and shedding of the murine mammary tumor virus (MuMTV) glycoproteins and in virus production was investigated in a clonal mammary tumor cell line, GR-3A, using two inhibitors of protein glycosylation, 2-deoxyglucose (2-DG) and tunicamycin (TM). It was found that both 2-DG and TM completely inhibited the synthesis of the MuMTV envelope precursor polyprotein, Pr70env, and, as a consequence, the synthesis of the viral glycoproteins gp52 and gp36. By contrast, the synthesis of Pr73gag, the polyprotein precursor of the internal structural proteins of the virus, was only inhibited by 10-15% by 2-DG and TM. Although 2-DG and TM blocked the synthesis of Pr70env, a new polypeptide, related to gp52 and gp36, with a mol wt of 60,000 (P60env) was found to be synthesized in the treated cells. The P60env molecules appeared to be degraded intracellularly since they were not found to (1) undergo site-specific cleavage; (2) accumulate inside the cell or on the cell surface; (3) be secreted into the culture medium; and (4) be incorporated into the virions produced during the drug treatment. In spite of the lack of gp52 and gp36 synthesis in the presence of TM and 2-DG, mature MuMTV particles containing the characteristic surface projections known to be composed of gp52 and gp36 continued to be assembled and released at a reduced rate for at least 30 hr. In addition, the buoyant density and the polypeptide composition of the particles were found to be identical to virions produced by untreated cells. Thus, the virions assembled and released during 2-DG and TM treatment were not defective. Our investigations into the origin of gp52 and gp36 in these particles revealed that both molecules were synthesized prior to 2-DG and TM treatment and continued to be incorporated, along with the newly synthesized viral core proteins, into budding virions during the drug treatment. Furthermore, we found that gp52 and P75env (an aberrant form of Pr70env) that were not incorporated into virions continued to be shed normally from the cell during drug treatment. In conclusion, our results suggest that MuMTV assembly is not dependent on the synchronized synthesis of the viral core and envelope polypeptides, and that the assembled virions contain the correct ratio of these polypeptides, even when their ratio in the cell varies.