Biology of Mammary Tumor Viruses ¹

Robert D. Cardiff
Bruce W. Altrock

Department of Pathology
School of Medicine
University of California
Davis, California

Mouse mammary tumors are the result of interactions between 1) a tumor virus, 2) a genetically susceptible host, 3) hormones, 4) the hosts' immunological responsiveness, and 5) other factors. The understanding of this complex system has been enriched by contributions from many disciplines including virology, genetics, endocrinology, and immunology. Advances in these fields have complemented one another when applied to the mouse mammary tumor model. As a result, the mouse mammary tumor system has become a prototype for research in solid tumors. This review is designed to alert the readers to the current concepts of the relationship of one factor, the mouse mammary tumor virus (MMTV), to the others involved in mouse mammary tumor biology. It ends on a speculative theme anticipating future developments in the field.

Early studies of mouse mammary tumors emphasized hereditary factors. Inbred strains of mice were, in part, developed to study the genetics of this and other neoplasms. As a result of the development of these strains, investigators in Europe and the United States were able to study the genetics of low and high breast cancer strains of mice. These experiments culminated in the recognition by the staff of The Jackson Laboratories and by Korteweg in the Netherlands of an extrachromosomal factor causing mammary adenocarcinomas in mice ( 1, 2). Shortly thereafter, Bittner discovered that this factor was transmitted via the milk of high tumor incidence mothers to their offspring ( 3). This factor became known as the Bittner agent, milk agent, mouse mammary tumor agent, and, following morphological and biophysical characterization ( 4, 5), the mouse mammary tumor virus.

The mouse mammary tumor virus is similar in many respects to other RNA tumor viruses ( 6). As with the other members of the Retraviridae family, MMTV has a high molecular weight polyploid RNA genome ( 7, 8) which is associated with the characteristic Retraviridae enzyme RNA-dependent DNA polymerase and is located within the viral core ( 9). The core is surrounded by a lipid envelope which has external projections or spikes. The distinctive eccentric location of the core within this virus led Bernhard to call the mature mouse mammary tumor virion "B-particle" to distinguish its structural arrangement from the C-type retravirus particles ( 10). The MMTV core apparently arises from a precursor, the intracytoplasmic A-particle ( 11, 12), which contains RNA-dependent DNA polymerase and the internal viral polypeptides (p28, p14 and p10) in precursor form ( 12, 13). The virion buds from the surface of infected cells where it acquires its lipid envelope and the surface projections ( 6, 14). These projections are thought to be composed of two major glycoproteins with molecular weights of 52,000 and 36,000 daltons ( 15, 16). The mouse mammary tumor virus is the prototype for the type B oncornaviruses because of its biophysical, biochemical, and morphological characteristics and because of its tumorigenic capabilities ( 17).

The genetic information of MMTV can be found in two phases, RNA and DNA ( 18). The DNA phase is integrated into the chromosomal DNA of all mice thus far studied and can be transmitted genetically in this form (an endogenous viral sequence) ( 19). In most strains of mice, these endogenous or germinal DNA sequences are not usually expressed and reports of their recovery as mature virions are rare ( 6). The GR mouse strain has been an interesting exception in which the germinal DNA sequences are expressed ( 20, 21).

The B-particle (exogenous virus) contains the RNA phase and is transmitted extracellularly via the milk in most mouse strains ( 22). Although B-particles, infectious agents and/or viral antigens have been reported in a variety of organs, the exogenous particle appears to primarily infect the mammary gland ( 23). The exogenous virus usually becomes integrated into the DNA of the infected mammary cells and can be passed in this manner to subsequent generations of daughter cells ( 24). These events are associated with mammary tumorigenesis. Mammary tissue thus has been the focus of MMTV research.

Although the emphasis has been on the virus in mammary adenocarcinoma, the reader is reminded that the traditional point of view envisions mammary cancer as a multifactorial disease. This point of view, most effectively espoused by Dr. Walter Heston ( 25), emphasizes genetic, hormone, and viral cofactors. The theoretical modes of action of these cofactors were expanded by the description of an intermediate preneoplastic tissue (the hyperplastic alveolar nodule) ( 26, 27).

Genetics. Although early mouse mammary tumor studies concerned the hereditary aspect of cancer, the discovery of the viral milk agent tended to reduce the emphasis on genetic factors ( 25). Fortunately, studies of genetic susceptibility to mammary cancer continued and later complemented the virological work ( 6, 25).

In 1965, Mühlbock reaffirmed male transmission of mammary cancer in the relatively new European mouse strain, GR ( 30). In 1968, Bentvelzen, through a study of the GR strain, came to the then startling conclusion that the mouse mammary tumor virus was transmitted by germ cells of the GR mouse ( 21). Thus, passage of the high-risk agent to offspring was not limited to transmission through the mother's milk.

Subsequently the Dutch investigators found, using GRxC57BL crosses, that tumor development followed a simple Mendelian ratio ( 21, 31). They proposed that mouse mammary tumorigenesis was controlled by a single gene ( 32). This concept was extended to define a single gene locus ( MTV-2) controlling the expression of MMTV in the milk of GR mice. A congenic GR strain lacking the MTV-2 gene has been described which has a low tumor incidence ( 33).

The single gene hypothesis has been disputed by others. Nandi and Helmich interpreted their data to mean that at least two genes were involved in mammary tumorigenesis ( 34). In more extensive experiments using C57BLxGR backcrosses, Heston and Parks concluded that the tumor incidence in the second backcross generation was inconsistent with a single gene hypothesis ( 25, 36).

The correct interpretation of these various experiments has been hotly debated ( 36). All appear to agree, however, that a limited number of genes influence viral expression and tumorigenesis. Implicit in most interpretations has been the idea that genetic factors and virus expression were interrelated in the control of tumor incidence and latency. These factors are further complicated by the numerous host factors which exert control on the various phases of the viral infection. For example, it has been observed that susceptibility to a given virus stock varies with the test mouse strain used ( 37, 38, 39). Bentvelzen has reviewed a number of potential host factors which may influence viral infection and neoplastic transformation ( 6, 30).

Immunology. One prominent host factor capable of influencing MMTV infection and tumorigenesis is the host's immunological responsiveness to MMTV-related antigens. Our understanding of the immunology of MMTV has been considerably extended in the last few years but is still far from complete. The current data indicate that the quality as well as quantity of an animal's response to MMTV may be important. An inappropriate immune response to MMTV may be ineffective or, worse yet, may actually favor the genesis and/or growth of an MMTV-induced tumor.

Early immunological studies used tissue transplantation techniques to investigate mammary tumor antigenicity. The predominant antigenicity of MMTV-induced tumors showed little variation from tumor to tumor and was attributable to the presence of exogenous viral antigens ( 40, 46). Non-viral non-cross-reacting mammary tumor antigens were reported ( 47, 48, 49, 50, 51), but these were clearly much less immunogenic than the viral antigens.

In initial studies, mice infected with exogenous MMTV as neonates appeared to be tolerant to viral-associated antigens ( 43). These neonatally infected animals did not exhibit a detectable anti-viral immunity of their own and apparently could not be immunized against challenge with transplanted tumor cells. Blair et al. ( 52) later reported the detection of serum antibodies against MMTV-associated antigens following immunization of infected mice. This report was confirmed and extended by other investigators ( 53, 54, 55).

With the development of sensitive, reproducible means of assessing cell-mediated immunity in vitro came the observation that mice infected with exogenous MMTV exhibit a naturally-occurring cellular response to MMTV-induced tumor cells ( 47, 56). Serological factors were later described in these mice which inhibited (blocking factors) or otherwise affected the ability of lymphocytes (from infected mice) to kill mammary tumor cells in vitro ( 48, 57, 58).

Serum from exogenously infected mice has been shown to contain antibodies directed against MMTV antigens ( 59, 60, 61, 62). In addition, MMTV antigens have also been found in these sera ( 63). Serum MMTV antigens may combine with circulating anti-MMTV antibodies and neutralize their effectiveness by forming immune complexes which then accumulate in the glomeruli of the kidney ( 60).

Strains of mice which carry only the endogenous form of MMTV can be successfully immunized against MMTV and protected against the challenge with transplanted mammary tumor cells ( 40, 41, 42, 43, 44, 45, 46). These mice respond to MMTV-associated antigens with the production of humoral and cellular immune responses which, unlike responses seen in exogenously infected mice, are effective against MMTV or mammary tumor tissue.

Despite the antigenicity of MMTV-infected tissues, and despite the presence of both cellular and humoral responses to viral antigens, mice infected with exogenous MMTV develop mammary tumors. Attempts to augment the immune responsiveness of exogenously infected mice to viral antigens specifically ( 53, 64, 65, 66, 67, 68, 69) or to generally stimulate their immune systems ( 43) have proven ineffective in protecting them and frequently have accelerated mammary tumor development. Immunosuppressive measures, on the other hand, have been shown to improve an infected host's ability to resist MMTV-induced tumorigenesis ( 70, 71, 72, 73, 74). Uninfected mice can be successfully immunized in such a way as to resist subsequent challenge with tumor cells ( 75). A host's response to MMTV and MMTV-induced tumor cells may or may not be effective in protecting the animal from mammary tumors, but clearly influences the course of events in MMTV infection and tumorigenesis. The quality, quantity, and timing of an animal's anti-MMTV response appear to be the critical factors in determining its effectiveness.

Hormones. The endocrine status of the mouse has long been recognized to play an important role in mouse mammary tumorigenesis ( 76, 77, 78, 79). Classical endocrine organ ablation and hormonal replacement experiments have demonstrated hormone control of this system and have emphasized the role of lactogenic hormones (i.e., prolactin) and estrogen ( 77). Increasing the number of pregnancies also increases the risk of mouse mammary tumorigenesis ( 14, 78, 79). Nulliparous mice of some strains show a lower tumor incidence than their multiparous counterparts ( 14, 80, 81, 82, 83). Many early workers thought that the increased tumor incidence associated with hormonal stimulation was related to increased levels of viral expression ( 14).

With the development of in vitro systems, the role of hormones could be systematically studied. McGrath first demonstrated that glucocorticoids (e.g., cortisol, hydrocortisone) induce viral expression in primary cultures of mammary tumor cells ( 84). Subsequently, glucocorticoid induction of viral expression has been demonstrated in long-term primary cell cultures ( 85), in several established mouse mammary tumor lines ( 86, 87, 88), and in heterologous MMTV-infected cells ( 88, 89). MMTV has been found to be inducible by a number of glucocorticoids, but the most effective steroid has been the synthetic hormone dexamethasone ( 86, 87, 90). Dexamethasone induction of MMTV requires the appropriate hormone receptors ( 14, 87) and has been shown to induce an almost instantaneously increased rate of MMTV RNA synthesis ( 91). This increased RNA synthesis did not require DNA or protein synthesis ( 91). The hormone effects have been shown to be dose dependent ( 91).

Synergistic interactions have been described between insulin and hydrocortisone ( 84), insulin, hydrocortisone and estradiol ( 14, 87), and insulin and prolactin ( 92). These effects have not had the full benefit of molecular analysis. As a result, the exact mechanism and full significance of hormonal action and interaction in this system remain unclear. It does appear, however, that the same hormones which produce mammary tumors in the animal also stimulate virus production in vitro ( 14, 87). It remains to be seen whether this is a primary effect on viral synthesis or merely reflects optimal tissue culture conditions.

Recent demonstrations of infection of heterologous (feline kidney, rat hepatoma, and mink lung) cells in vitro with MMTV have created important new avenues of research ( 89). Heterologous cell infection has already provided new insight into the hormonal regulation of virus expression, into the antigenic structure of the virus, and into the molecular biology of the virus. It will make the study of virus infection and replication possible in previously uninfected cells and lead the way to genetic experiments.

Virus. The basic, but often unstated, assumption in many studies in the mouse mammary tumor system is that virus expression is related to tumorigenesis. This assumption is consistent with the widely accepted oncogene hypothesis which was developed for the endogenous C-type viruses ( 93) and has been modified to fit the murine mammary tumor virus system ( 6, 94, 95).

Investigators have long related titers of virus in the milk of various strains to the mammary tumor incidence of those strains ( 14). Numerous studies involving virus titration curves have attempted to relate the amount of virus which a group of mice receive to the ultimate tumor or nodule incidence in that group ( 14, 96, 97, 98, 99, 100, 101). More recently, the development of more sensitive quantitative assays has reinforced these impressions. For example, nucleic acid hybridization studies showed that the highest levels of MMTV RNA resided in the organs of those strains of mice having the highest tumor incidence ( 102). Others, using radioimmunoassays, have related the high levels of viral antigens in the milk of a given strain to the tendency of that strain to develop cancer ( 103, 104).

An example of the quantitative relationship between virus and tumorigenesis has come from our studies of the inbred mouse strain BALB/c (tumor incidence less than 1%) and the virus-infected subline BALB/cfC3H (tumor incidence of 87% at 16 months of age). BALB/c females have no detectable MMTV antigens in their milk and their BALB/c lactating mammary glands contain no detectable MMTV by electron microscopy, by immunoperoxidase, or by radioimmunoassay ( 62, 105). The BALB/c normal organs and lactating mammary gland contain approximately seven endogenous MMTV gene copies per diploid BALB/c cell. MMTV RNA can be detected in the BALB/c cytoplasmic extracts ( 106).

Our BALB/cfC3H colony was established at the Simonsen Laboratories, Gilroy, California (through the auspices of the Drug Evaluation Branch, National Cancer Institute), by fostering nursing BALB/c pups on milk agent-bearing C3H/He mothers. Once infected, the subsequent generations of BALB/cfC3H females were nursed on their own mothers. With a tumor incidence of 87%, the colony contains a relatively high proportion of animals which can be expected to develop tumors (high risk) and a few which will not (low risk).

As one possible parameter to define tumor risk before tumor development, MMTV concentrations in the milk of individual BALB/cfC3H animals were determined by radioimmunoassay ( 107) and the animals were held for tumor development. The animals segregated cleanly into two groups. All animals which developed tumors by 16 months of age had high levels of MMTV in their milk by the second lactation. The animals with low levels of virus in their milk did not develop tumors by the time the experiment was terminated at 16 months. The 95% confidence intervals for viral expression levels in these two groups of mice did not overlap.

The same pattern of expression was observed in a large group of BALB/c animals which were experimentally injected with one of seven log dilutions of MMTV at 4-5 weeks of age ( 107). Those injected animals which developed tumors by 16 months of age previously expressed high levels of MMTV in their milk by the third lactation while those which did not develop tumors expressed, at best, very low levels of viral antigens ( 107). In general, more mice in high dosage groups showed the high viral expression levels indicative of high risk of tumor development. The development of high virus levels was, however, an all or none phenomenon in that those animals in the high dosage groups which did not produce high levels of the virus did not develop tumors.

The relationship between input dose of virus, subsequent infection and tumorigenesis was observed by calculating the 50% infectious dose (ID₅₀) and 50% tumor dose (TD₅₀) of MMTV ( 107, 108). The major effect of injecting mice with lower doses of MMTV was to increase the latency period prior to detectable milk virus expression. That is, the calculated ID₅₀ for infection by the first lactation was approximately 19 millimicrograms/mouse (50% of the mice given this dose would be expected to have MMTV positive milk at the first lactation, but by the seventh pregnancy, most of the animals, regardless of the dose of MMTV received, expressed MMTV milk antigens and were scored as infected. The calculated ID₅₀, therefore, dropped to 0.01 millimicrogram/mouse or about 20,000 particles per mouse when milk from later lactations was examined.

Tumorigenesis was also dose dependent. The calculated TD₅₀ at 16 months of age with all but one of the "high risk" high expressor animals developing tumors was 24 millimicrogram/mouse ( 107). A comparison of the infectious dose and tumor dose data reveals that a four log difference exists between the final ID₅₀ and the TD₅₀. Thus, while MMTV in this context is necessary for tumorigenesis, there are many host-virus interaction which do not result in tumors.

The role of high MMTV input and high MMTV expression in the high-risk animal is not understood at a cellular level, but the immunoperoxidase technique provides one means of examining the cellular events involved. Almost all BALB/cfC3H tumor cells express high levels of MMTV antigens when examined by immunoperoxidase ( 105). In contrast, the BALB/cfC3H lactating mammary glad consists of small clusters of mammary epithelial cells which are positive for MMTV antigens and many cells which do not express any viral antigens. If one assumes (an unproven assumption) that only those cells expressing antigen are infected with exogenous virus, then the mammary epithelium is heterogeneous, consisting of infected and uninfected cells. Since the hyperplastic alveolar nodules all express viral antigen, it is also reasonable to assume that the nodule cells are derived from those mammary epithelial cells which express MMTV antigens. The viral antigen expressing cells could be the only cells at risk of undergoing nodulogenesis and/or tumorigenesis.

Molecular hybridization studies also suggest that tumors emerge from a select subpopulation. Morris et al. ( 109) demonstrated that non-mammary organs in most mouse strains contain five to nine copies of MMTV DNA per diploid genome as calculated from nucleic acid hybridization kinetic analysis. Mammary tumors, however, contain as many as 30 copies of MMTV DNA per diploid genome. The additional MMTV copies in tumors were interpreted to imply infection with exogenous MMTV.

Using the same techniques, V. Morris, H. Varmus and C. Cohen in collaboration with our laboratory found that BALB/c and BALB/cfC3H normal non-mammary organs from our colony have approximately seven copies of MMTV DNA per diploid genome. The BALB/cfC3H tumors have a variable MMTV copy number up to 44 copies per cell. The lactating mammary gland might be expected to contain an intermediate MMTV copy number but primiparous and multiparous normal lactating mammary glands had the same number of MMTV copies as the non-mammary organs, i.e., seven copies per diploid genome.

Since tumors contain more MMTV DNA than the normal lactating mammary gland, the viral DNA copies may have increased during the tumorigenic process, or the tumor cells may arise from a subpopulation of cells with a high MMTV copy number. Drs. Schlom and Drohan at the National Cancer Institute, using their "tumor-specific" MMTV probe, found high levels of tumor-specific sequences in BALB/cfC3H tumors but lower levels in the BALB/cfC3H lactating mammary gland ( 110). As a result of reconstruction experiments, the lactating mammary gland was estimated to have less than one tumor-specific MMTV DNA per cell. Slightly higher estimates were obtained from reconstruction restriction endonuclease experiments ( 111).

Simple arithmetic tells us that an average of one copy per cell could also be ten copies in one out of every ten cells. This arrangement would then be consistent with the clusters of cells observed in the immunoperoxidase studies. The data are all consistent with the notion that BALB/cfC3H mammary tumors arise from a subpopulation of cells. The ultimate form of such a subpopulation is a monoclonal proliferation.

The clonal origin of a number of other spontaneous and experimental tumors has been well documented ( 112). Polyclonal tumors, on the other hand, occur less frequently. Previous studies by Mintz using allophenic chimeras ( 113, 114) and by Hilgers on hormone-sensitive tumors ( 115) have suggested a clonal origin for mouse mammary tumors. Recent experiments using restriction endonuclease mapping have also implied that mouse mammary tumors are monoclonal ( 116, 117).

Support for the monoclonal origin of tumors in other systems has been based on three types of observations: 1) sex-linked polymorphic isoenzymes such as glucose-6-phosphate dehydrogenase in heterozygous females indicate that all tumor cells originate from a single cell ( 112), 2) immunoglobulin produced by myelomas or B cell lymphomas tend to be of a single type, indicating clonal origin ( 119), and 3)cytogenetic studies indicate that some tumors have a uniform karyotypic aberration, such as the Philadelphia chromosome in acute myelogenous leukemia ( 118, 119). Unfortunately, such marker systems which would be useful in the study of mouse mammary tumors have not yet been developed in inbred strains of mice. The clonal hypothesis in mouse mammary tumors awaits a technology which can test its validity.

If independently verified, a monoclonal origin for mouse mammary tumors would mean that they originate from a single somatic cell. This would have a number of important biological implications which can simply be paraphrased from Boveri ( 120): 1) the initial transformation event is rare, 2) the critical event is far removed from the final phenotypic expression of the cancer cell, and 3) virus infection is not in and of itself sufficient for tumorigenesis. As such, MMTV can be seen in its proper perspective as a cofactor in neoplasia since mammary neoplasia requires a series of events ( 121). As these events are unraveled with the powerful tools now available, a new chapter in the biology of the mouse mammary virus will be written.

The authors are indebted to Dr. Harold Varmus, Dr. Craig Cohen, and Dr. Vincent Morris of the University of California, San Francisco, and Dr. Jeffrey Schlom and Dr. William Drohan of the National Cancer Institute for sharing preprints of their work, permitting discussion of our unpublished data, and for their interest in our joint ventures and their many hours of discussion. The authors wish to acknowledge the work of Dr. L.R. Ayyagari and Ms. Judith St. George which has contributed to the concepts discussed herein and to express their appreciation to Ms. J. K. Lund for her editorial assistance.

¹This work was supported in part by NIH-NCI contract NO1 CP61013 within the Virus Cancer Program, by NIH grant 21454, and PHS training grant 05245.

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