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Amplification and overexpression of erbB2/neuproto-oncogene is observed in 20–30% human breast cancer and is inversely correlated with the survival of the patient. Despite this, somatic activating mutations within erbB2in human breast cancers are rare. However, we have previously reported that a splice isoform of erbB2, containing an in-frame deletion of exon 16 (herein referred to as ErbB2ΔEx16), results in oncogenic activation of erbB2because of constitutive dimerization of the ErbB2 receptor. Here, we demonstrate that the ErbB2ΔEx16 is a major oncogenic driver in breast cancer that constitutively signals from the cell surface. We further show that inducible expression of the ErbB2ΔEx16 variant in mammary gland of transgenic mice results in the rapid development of metastatic multifocal mammary tumors. Genetic and biochemical characterization of the ErbB2ΔEx16-derived mammary tumors exhibit several unique features that distinguish this model from the conventional ErbB2 ones expressing the erbB2proto-oncogene in mammary epithelium. Unlike the wild-type ErbB2-derived tumors that express luminal keratins, ErbB2ΔEx16-derived tumors exhibit high degree of intratumoral heterogeneity co-expressing both basal and luminal keratins. Consistent with these distinct pathological features, the ErbB2ΔEx16 tumors exhibit distinct signaling and gene expression profiles that correlate with activation of number of key transcription factors implicated in breast cancer metastasis and cancer stem cell renewal.

Animal models of prostate cancer have been limited in number and in relevance to the human disease. With the advancement of transgenic and knockout technologies, combined with tissue specific promoters and tissue-specific gene ablation, a new generation of mouse models has emerged. This review will discuss various animal models and their inherent strengths and weaknesses. A primary emphasis is placed on mouse models that have been designed on the basis of genetic alterations that are frequently found in human prostate cancer. These models display slow, temporal development of increasingly severe histopathologic lesions, which are remarkably restricted to the prostate gland, a property similar to the ageing related progression of this disease in humans. The preneoplastic lesions, akin to what is considered as prostatic intraepithelial neoplasia, are consistent major phenotypes in the models, and, therefore, are discussed for histopathologic criteria that may distinguish their progressions or grades. Finally, considering that prostate cancer is a complex multifocal disease, which is likely to require multiple genetic / epigenetic alterations, many of these models have already been intercrossed to derive mice with compound genetic alterations. It is predicted that these and subsequent compound mutant mice should represent “natural” animal models for investigating the mechanism of development of human prostate diseases, as well as, for preclinical models for testing therapeutics.

In aging men, the prostate gland becomes hyperproliferative and displays a propensity toward carcinoma. Although this hyperproliferative process has been proposed to represent an inappropriate reactivation of an embryonic differentiation program, the regulatory genes responsible for normal prostate development and function are largely undefined. Here we show that the murine Nkx3.1 homeobox gene is the earliest known marker of prostate epithelium during embryogenesis and is subsequently expressed at all stages of prostate differentiation in vivo as well as in tissue recombinants. A null mutation for Nkx3.1 obtained by targeted gene disruption results in defects in prostate ductal morphogenesis and secretory protein production. Notably, Nkx3.1 mutant mice display prostatic epithelial hyperplasia and dysplasia that increases in severity with age. This epithelial hyperplasia and dysplasia also occurs in heterozygous mice, indicating haploinsufficiency for this phenotype. Because human NKX3.1 is known to map to a prostate cancer hot spot, we propose that NKX3.1 is a prostate-specific tumor suppressor gene and that loss of a single allele may predispose to prostate carcinogenesis. The Nkx3.1 mutant mice provide a unique animal model for examining the relationship between normal prostate differentiation and early stages of prostate carcinogenesis.

Genomic DNAs from dimethylbenzanthracene-induced BALB/c mouse mammary tumors arising from the transplantable hyperplastic outgrowth (HPO) line designated DI/UCD transformed NIH 3T3 cells upon transfection. Transforming activity was attributed to the presence of activated Harvey ras-1 oncogenes containing an A----T transversion at the middle adenosine nucleotide in codon 61. DNAs from untreated DI/UCD HPO cells and radiation-induced and spontaneous mammary tumors from the DI/UCD HPO line failed to transform NIH 3T3 cells. The results indicated that the mutation activation of Harvey ras-1 oncogenes was specific to dimethylbenzanthracene treatment in the mouse mammary tumor system.

Antigenic determinants of mouse mammary tumor virus (MuMTV) from the low-mammary-tumor-incidence strain BALB/cNIV were compared by competition radioimmunoassay with those of MuMTV's isolated from several high- and low-mammary-tumor-incidence mouse strains, using rabbit hyperimmune sera against BALB/cNIV MuMTV and against MuMTV from the high-mammary-tumor-incidence strain BALB/cfC3H. Using anti-BALB/cfC3H serum in competition radioimmunoassay, BALB/cNIV MuMTV lacked antigenic determinants present on MuMTV's from the BALB/cfC3H, C3H, and GR strains. With anti-BALB/cNIV serum in competition radioimmunoassay, type-specific antigenicity was detected with BALB/cNIV MuMTV. We found class-specific antigenicities on BALB/cNIV MuMTV that were shared with RIII and C3Hf MuMTV's BALB/cNIV MuMTV was reportedly derived from the C3Hf strain by infection of BALB/c mouse mammary tissue after transplantation into a (BALB/c X C3Hf)F1 hybrid followed by retransplantation into the BALB/c strain (D. R. Pitelka, K. B. DeOme, and H. A. Bern. Proc. Am Assoc. Cancer Res. 6:51, 1965). However, BALB/cNIV MuMTV contained type-specific antigens not present on C3Hf MuMTV. The possible origin of these determinants is discussed.

Since the detection of abnormal DNA content in the cells of transitional cell carcinoma can be obscured by the presence in the population of diploid stromal and inflammatory cells, it would be desirable to have a second flow cytometric parameter to complement DNA analysis. The specific identification of carcinoma cells using monoclonal antibodies directed against epithelial intermediate filament proteins (cytokeratins) would provide a method with which to identify the transitional cells in solid tumors, voided urines or bladder washings. As a preliminary study we have tested cell-free urine samples using a double antibody radiometric assay (IRMAK) for cytokeratins released from transitional cells. The additional information from cytokeratin analyses increased the specificity of disease detection.

Experimental carcinogenesis has led to a concept that defines two discrete stages in the development of skin tumors: (i) initiation, which is accomplished by using a mutagen that presumably activates a protooncogene, and (ii) promotion, which is a reversible process brought about most commonly by repeated application of phorbol esters. We have created a transgenic mouse strain that carries the activated v-Ha-ras oncogene fused to the promoter of the mouse embryonic alpha-like, zeta-globin gene. Unexpectedly, these animals developed papillomas at areas of epidermal abrasion and, because abrasion can also serve as a tumor-promoting event in mutagen-treated mouse skin, we tested these mice for their ability to respond to phorbol ester application. Within 6 weeks virtually all treated carrier mice had developed multiple papillomas, some of which went on to develop squamous cell carcinomas and, more frequently, underlying sarcomas. We conclude that the oncogene "preinitiates" carrier mice, replacing the initiation/mutagenesis step and immediately sensitizing them to the action of tumor promoters. In addition, treatment of the mice with retinoic acid dramatically delays, reduces, and often completely inhibits the appearance of promoter-induced papillomas. This strain has use in screening tumor promoters and for assessing antitumor and antiproliferative agents.

The distribution of dengue-2 antigens was studied in infected monkey kidney cells (LLC MK2) using an indirect, horseradish peroxidase-conjugated immunoglobulin technique. This procedure allowed both light and electron microscopic examination of serial-step sections of individual cells cut in a plane perpendicular to the monolayer. Both virion and nonvirion antigens were identified on the plasma membrane with this technique. A diffuse cytoplasmic reaction product was also present. The intensity and distribution of the cytoplasmic reaction product was related to disruption of the plasma membrane.

The effect of mammary gland-specific expression of the polyomavirus middle T antigen was examined by establishing lines of transgenic mice that carry the middle T oncogene under the transcriptional control of the mouse mammary tumor virus promoter/enhancer. By contrast to most transgenic strains carrying activated oncogenes, expression of polyomavirus middle T antigen resulted in the widespread transformation of the mammary epithelium and the rapid production of multifocal mammary adenocarcinomas. Interestingly, the majority of the tumor-bearing transgenic mice developed secondary metastatic tumors in the lung. Taken together, these results suggest that middle T antigen acts as a potent oncogene in the mammary epithelium and that cells that express it possess an enhanced metastatic potential.

Mouse mammary tumor virus (MMTV)-induced mammary adenocarcinomas can develop from several different premalignant precursors common in GR mice. Insertion mutagenesis of the mammary protooncogenes int-1 and int-2 was studied in this multistep system by analyzing samples from various stages of neoplastic development for novel int-1 and int-2 restriction fragments generated by MMTV provirus integration. int-1 and int-2 insertion mutations were observed in both premalignant lesions and malignant tumors. Some of the tumors with insertion mutations were experimentally derived from insertion mutation-free premalignant precursors. Each class of neoplasm examined had a characteristic frequency of int-1 and int-2 insertion mutations; however, no correspondence was observed between neoplasm morphology and mutation of either gene. These results indicate that insertion mutation of the int-1 and int-2 loci by MMTV provirus can be involved in the earliest identifiable stages of neoplastic development as well as during progression of premalignant lesions to tumors. Insertion mutation of int-1 and int-2 is therefore not stage specific in this system.

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.

The methylation state of endogenous mouse mammary tumor virus (MuMTV) proviral DNA was examined in normal mouse tissues. DNAs from various tissues were cleaved with the methylation-sensitive enzymes HhaI and HpaII and analyzed by Southern blotting. Tissue-specific MuMTV proviral DNA methylation patterns were found in the BALB/c, C3H, C57BL, GR/A, and GR-Mtv-2- mouse strains. MuMTV proviral DNA was hypomethylated in DNAs from the spleens and testes of all strains examined. The GR/A mouse strain, which was most thoroughly studied, also showed hypomethylation of MuMTV proviral DNA in bone marrow and placental tissues. Analysis of RNAs extracted from GR/A liver, mammary tumor, testes, placenta, and spleen tissues demonstrated that MuMTV proviral hypomethylation need not reflect significant proviral transcription.

We examined the genetic structure, in terms of restriction endonuclease recognition sites, of the milk-transmitted, low-oncogenic mouse mammary tumor virus (MuMTV) of the BALB/cNIV mouse strain. An analysis with EcoRI documented the presence of acquired cNIV proviruses in the mammary tumor DNAs of BALB/cNIV animals. A comparison of tumor DNAs digested with PstI showed that both the cNIV MuMTV and C3Hf MuMTV proviruses lacked the 4.3- and 1.1-kilobase pair fragments characteristic of C3H MuMTV patterns. An examination of mammary tumor and normal, nonmammary tissue DNAs with BamHI supported the idea that the cNIV MuMTV is identical to the C3Hf MuMTV and demonstrated that these two low-oncogenic proviruses are identical to the high-oncogenic C3H MuMTV provirus with respect to a pair of BamHI sites which define a 1.3-kilobase pair fragment. For each of the three MuMTV strains, we also mapped DNAs generated in isolated virions by reverse transcription of their genomic RNAs. Our results showed that cNIV and C3Hf MuMTV are distinct entities by virtue of an additional PstI site within the cNIV long terminal repeat sequence. Another unique feature of cNIV MuMTV revealed by the analysis of virion-generated DNAs was the existence of a family of genomes within the cNIV population. We concluded that cNIV is distinct from its presumptive C3Hf MuMTV predecessor.

The methylation and amplification of mouse mammary tumor virus (MuMTV) proviral DNA was investigated in normal, premalignant, and malignant tissues of GR/A mice. The proviral methylation pattern was examined with the restriction enzyme HhaI, which fails to cleave methylated DNA. MuMTV proviral DNA from liver, kidney, and heart was highly methylated. Proviral DNA was somewhat undermethylated in mammary gland cells from virgin and lactating mice and extensively undermethylated in cells from premalignant outgrowths, pregnancy-dependent tumors, and pregnancy-independent tumors. The restriction enzyme SacI was used to detect additional proviruses in the same cells. No additional proviral copies of MuMTV were detected in liver, kidney, or heart cells or in mammary gland cells from virgin mice. Some mammary gland cells from lactating mice appeared to contain additional copies of the endogenous, highly oncogenic GT-MTV-2 provirus. Premalignant outgrowth, pregnancy-dependent tumor, and pregnancy-independent tumor cells contained an average of two to three additional copies per cell of the GT-MTV-2 provirus. Thus, neoplasia in GR/A mice was directly associated with quantized increases in MuMTV proviral DNA undermethylation and GR-MTV-2 proviral DNA amplification. Restriction enzyme analysis suggested that premalignant outgrowths and pregnancy-dependent tumors both consisted largely of heterogenous cell populations, although some evidence of clonal dominance was detected.

Expression of the activated neu oncogene in transgenic mice has been associated with both the synchronous (single-step) and the stochastic (multistep) transformation of the mammary epithelium. To determine the basis for these conflicting observations, additional strains of transgenic mice carrying the activated neu oncogene under the transcriptional control of the mouse mammary tumor virus promoter/enhancer were produced. Activated neu transgene expression, as measured by in situ hybridization and ribonuclease protection assays, resulted in rapid conversion of the normal mammary epithelium to malignant phenotype in three independent strains of mice. Expression of the transgene in male mice led to epithelial hyperplasia of the epididymis and male infertility but not malignancy. These results indicate that tissue context is an important parameter in malignant progression and that expression of appropriate levels of activated neu is sufficient for rapid production of mammary tumors in transgenic mice.

Activation of the c-Src tyrosine kinase has been implicated as an important step in the induction of mammary tumors in both mice and humans. To directly assess the effect of mammary gland-specific expression of activated c-Src, we established transgenic mice that carry a constitutively activated form of c-src under transcriptional control of the murine mammary tumor virus long terminal repeat. Female mice derived from several independent transgenic lines lactate poorly as a consequence of an impairment in normal mammary epithelial development. In addition to this lactation defect, female mice frequently develop mammary epithelial hyperplasias, which occasionally progress to frank neoplasias. Taken together, these observations suggest that expression of activated c-Src in the mammary epithelium of transgenic mice is not sufficient for induction of mammary tumors.

Studies of mouse mammary tumor virus (MMTV) have been impeded by the lack of an in vitro infectivity assay. We have developed a rapid, quantitative in vitro assay for MMTV infectivity based on the detection of positively staining foci by immunoperoxidase. This assay and a 50% end-point titration of MMTV infectivity gave identical virus titers. Infection of a rat hepatoma cell line, a feline kidney cell line, and a normal murine mammary gland cell line by virus from the mouse mammary tumor GR3A cell line was linear with respect to virus concentration. The infectious titers obtained in both homologous and heterologous cell lines were not significantly different, demonstrating a lack of host range specificity. Virus infectivity was inactivated by heating at 55 degrees C and by ultraviolet irradiation. Rabbit anti-MMTV serum neutralized the infectivity with a 50% neutralization end point of 1:5000. Applications of this assay to the study of the immunological, biological, and biochemical characteristics of MMTV are discussed.

The GR laboratory mouse strain has five endogenous proviral copies of the mouse mammary tumor virus (MMTV). One of these, Mtv-2, is unique because it causes mammary carcinomas in virtually 100% of breeding GR females prior to 1 year of age. To facilitate studies of this locus in particular, and mammary tumorigenesis in general, we genetically tailored a new mouse line, WXG-2, which bears Mtv-2 as its only endogenous MMTV provirus. The WXG-2 line was constructed by making hybrids between the GR strain and a wild mouse line free of both endogenous and exogenous MMTV, backcrossing to the MMTV-free line, and fixing the Mtv-2 locus in a population with the desired genotype. Mammary tumors were observed in 5 of the 20 hybrid females carrying the endogenous Mtv-2 provirus. The WXG-2 line represents a new model system for studying MMTV-induced mammary tumorigenesis in the absence of multiple endogenous proviruses.

Overexpression and amplification of the neu (c-erbB2, ERBB2) protooncogene have been implicated in the development of aggressive human breast cancer. To directly assess the effect of mammary gland-specific expression of the neu protooncogene, transgenic mice carrying unactivated neu under the transcriptional control of the mouse mammary tumor virus promoter/enhancer were established. By contrast to the rapid tumor progression observed in several transgenic strains carrying the activated neu transgene, expression of unactivated neu in the mammary epithelium resulted in the development of focal mammary tumors after long latency. The majority of the mammary tumors analyzed expressed elevated levels of neu-encoded mRNA and protein. Overexpression of neu in the mammary tumors was also associated with elevated neu intrinsic tyrosine kinase activity and the de novo tyrosine phosphorylation of several cellular proteins. Interestingly, many of the tumor-bearing transgenic mice developed secondary metastatic tumors in the lung. These observations suggest that overexpression of the unactivated neu protein can induce metastatic disease after long latency.

Transgenic mice expressing either the neu proto-oncogene or transforming growth factor (TGF-alpha) in the mammary epithelium develop spontaneous focal mammary tumors that occur after a long latency. Since the epidermal growth factor receptor (EGFR) and Neu are capable of forming heterodimers that are responsive to EGFR ligands such as TGF-alpha, we examined whether coexpression of TGF-alpha and Neu in mammary epithelium could cooperate to accelerate the onset of mammary tumors. To test this hypothesis, we interbred separate transgenic strains harboring either a mouse mammary tumor virus/TGF-alpha or a mouse mammary tumor virus/neu transgene to generate bitransgenic mice that coexpress TGF-alpha and neu in the mammary epithelium. Female mice coexpressing TGF-alpha and neu developed multifocal mammary tumors which arose after a significantly shorter latency period than either parental strain alone. The development of these mammary tumors was correlated with the tyrosine phosphorylation of Neu and the recruitment of c-Src to the Neu complex. Immunoprecipitation and immunoblot analyses with EGFR- and Neu-specific antisera, however, failed to detect physical complexes of these two receptors. Taken together, these observations suggest that Neu and TGF-alpha cooperate in mammary tumorigenesis through a mechanism involving Neu and EGFR transactivation.

A dominant restriction allele, Akvr-1r, from California wild mice (Mus musculus domesticus) confers resistance to exogenous ecotropic murine leukemia virus (MuLV) infection. The presence of an ecotropic MuLV envelope-related glycoprotein in uninfected virus-resistant cells suggests that viral interference is a possible mechanism for this resistance. We molecularly cloned the ecotropic MuLV envelope-related sequence from the genomic DNA of a wild mouse homozygous for the Akvr-1r locus. The cloned provirus was defective and contained a C-terminal end of the pol gene, a complete envelope gene, and a 3' long terminal repeat. The presence of this provirus was directly correlated with Akvr-1r-mediated virus resistance in cell cultures and hybrid mice. The Akvr-1r provirus restriction map and partial DNA sequence were identical to those of the Fv-4r allele, an ecotropic MuLV resistance locus from Japanese feral mice (M. musculus molossinus), which was previously shown to be allelic with the Akvr-1r gene. The 3' host flanking sequences of Fv-4r and Akvr-1r also had identical restriction maps. These findings indicate that Akvr-1r and Fv-4r are the same gene. It was probably acquired by interbreeding of these feral species in recent times. Conservation of this locus might be favored by the useful function that it performs in protection against ecotropic MuLV infection endemic in both populations of wild mice.

We used a two-dimensional Southern blotting procedure to analyze the tissue-specific methylation patterns of the five endogenous mouse mammary tumor viruses in the GR/A mouse strain. Our findings suggest that in certain tissues (brain, kidney, and liver) all proviruses are extensively methylated. In other tissues (spleen, placenta, and testes) all proviruses are hypomethylated to some degree. In these tissues individual proviruses display both quantitative and qualitative differences in methylation. We interpret the general patterns of tissue-specific hypomethylation in terms of a "hitch-hiker" model: mouse mammary tumor virus proviral methylation patterns reflect the tissue-specific activity of neighboring sequences. The observation that certain sites on particular proviruses are differentially methylated in a tissue-specific fashion may reflect tissue-specific differences in the makeup or conformation, or both, of proviral-containing chromatin.

We have examined an assortment of preneoplastic and neoplastic mouse mammary tissues for the presence of an mRNA which could encode the putative long terminal repeat gene product of mouse mammary tumor virus. We report here the detection of a novel mouse mammary tumor virus-specific, polyadenylic acid-containing transcript in certain preneoplastic and neoplastic mammary tissue of BALB/c mice. The molecule is 1.6 kilobases in length and contains sequences from the transcriptional leader and the U3 region of the proviral DNA. The upstream terminus of the 3' information lies 75 to 80 nucleotides from the beginning of the long terminal repeat open reading frame, in close proximity to a consensus splice acceptor in the DNA. The transcript was detected in hormonally or chemically induced neoplastic, preneoplastic, and lactating mammary tissue of BALB/c mice, but not in preneoplastic or tumor tissue induced by exogenous viruses in any strain of mice examined. This implies that the RNA we observed is transcribed from an endogenous provirus template.

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.

The polypeptide, antigenic, and morphological structure of the mouse mammary tumor virus was studied following protease digestion of intact virions. Intact, untreated virions (? = 1.17 g/ml) had characteristic envelope spikes, five major polypeptides, and were precipitated by antisera against gp52. Two of the major polypeptides, with molecular weights of 52,000 (gp52) and 36,000 (gp36), had carbohydrate moieties. Protease treatment resulted in spikeless, “bald” particles (? = 1.14 g/ml), which had altered surface antigenicity and which contained neither gp52 nor gp36. These data indicated that gp52 and gp36 were on the viral envelope. Bald particles retained a 28,000 dalton polypeptide (p28) which was proposed as the major internal polypeptide.

Immunofluorescence and electron microscopy were used to study the development and cytological distribution of dengue-2 virion and nonvirion antigens in monkey kidney cells. The type and combination of fixatives were found to affect the intensity of fluorescence. Paraformaldehyde fixation alone resulted in a low level of fluorescence, but an additional fixation step with ethanol or acetone resulted in maximum staining. No fluorescence was obtained after fixation with glutaraldehyde alone or in combination with other solvents. Methanol caused selective ablation of fluorescence by antibody to purified virions. Dengue-specific fluorescence that was most intense in the perinuclear area radiated in a granular pattern of decreasing intensity into the cytoplasm. The perinuclear fluorescence was associated with nonvirion antigens and the cytoplasmic fluorescence was associated with virion antigens. Electron micrographs of infected cells revealed vesicular bodies with reticular electron-dense centers, clusters of structurally complete virions deep within the endoplasmic reticulum, and single virus particles in vacuoles near the periphery of the cell.

In 10-day-old Balb/cCrgl mice, the subcutaneous injection of 0.1 µg of estradiol in distilled water per animal per day resulted in the conversion, over a 4 day period, of the original 3 cell layered cuboidal epithelium to a stratified, multilayered, fully keratinized epithelium. By light microscopy, there was development of a prominent stratum germinativum and of a mucinified surface on the 1st day, followed by the sequential formation of a stratum spinosum, a stratum granulosum, and a stratum corneum. By electron microscopy, the principal early modifications consisted of a marked increase in ribosomes, desmosomes, and 70 Å cytoplasmic filaments, the latter being aggregated into approximately 700 Å fibrils. The subsequent establishment of a keratin layer was preceded by the appearance of keratohyaline granules and the disappearance of mitochondria and endoplasmic reticulum in cells immediately above the stratum spinosum and by the development of a transitional cell layer in which there was progressive aggregation of cytoplasmic filaments and disappearance of nuclei, keratohyaline granules, and free ribosomes. In the upper stratum granulosum, transitional cell layer, and stratum corneum there were distinctive modifications in desmosomal structure (composite and modified desmosomes). The morphological and physiological significance of these observations is discussed.

We found that an endogenous mouse mammary tumor provirus, GR-MTV-8, is embedded within a member of the BAM HI family of long interspersed repetitive mouse DNAs. GR-MTV-8 appears to be transcriptionally silent at its normal chromosomal position in the mouse genome. The provirus is transcriptionally active, however, when cloned and transfected into mouse cells (Kennedy et al., Nature (London) 295:622-624, 1982). We propose that the transcriptional inactivity of GR-MTV-8 in situ is due to an inhibitory effect, possibly involving DNA methylation, attributable to the flanking BAM HI element.