New mammary epithelial and fibroblastic cell clones in coculture form structures competent to differentiate functionally.

We have established and characterized a spontaneously immortalized, nontumorigenic mouse mammary cell line, designated IM-2. IM-2 cells synthesize large amounts of the milk protein beta-casein upon addition of lactogenic hormones. The induction of beta-casein occurs rapidly and does not require any exogenous extracellular matrix components. The IM-2 cell line is morphologically heterogeneous and could be separated into cell clones with epithelial and fibroblastic characteristics. In monoculture, none of the epithelial clones could be induced to synthesize caseins. Coculture of epithelial and fibroblastic clones, however, rendered the epithelial cells competent to differentiate functionally; the addition of lactogenic hormones to these cocultures resulted in the synthesis of beta-casein in amounts comparable to that seen with the original IM-2 line. Using this unique cell system, we have investigated the interrelationships between different steps in differentiation leading to hormone-induced casein production. Independent of hormones, epithelial-fibroblastic cell contacts led to the formation of characteristic structures showing the deposition of laminin. We found that the epithelial cells located in these structures also exhibited significantly increased levels of cytokeratin intermediate filament polypeptides. Double immunofluorescence revealed that the cells inducible by hormones to synthesize casein, colocalized exactly with the areas of laminin deposition and with the cells showing greatly intensified cytokeratin expression. These results suggest that hormone-independent differentiation events take place in response to intercellular epithelial-mesenchymal contacts. These events in turn bring about a state of competence for functional differentiation after lactogenic hormonal stimulation.

Half-life of the Rous sarcoma virus transforming protein pp60src and its associated kinase activity.

The half-life of metabolically labeled pp60src of the Prague A strain of Rous sarcoma virus and of several transformation-defective, temperature-sensitive mutants was investigated by pulse-labeling infected cells with [35S]methionine, chasing for different times, and immunoprecipitating pp60src with tumor-bearing rabbit serum. These experiments showed that pp60src has a short half-life of approximately 60 min under normal physiological conditions and that the mutant pp60src proteins have similar half-lives to the wild type, irrespective of whether the cells are kept at the nonpermissive (42 degrees C) or permissive (35 degrees C) temperature. The half-life of the pp60src -associated kinase activity was determined by monitoring its decay by the immunoglobulin G heavy chain assay after the cells had been treated with several inhibitors of protein synthesis. In these experiments the kinase half-life was much longer than expected from the half-life of pp60src. The apparent contradiction between the half-lives of the kinase activity and the [35S]methionine-labeled pp60src protein could be resolved by the observation that treatment of cells with inhibitors of protein synthesis stabilized pp60src, resulting in a greatly extended half-life. Inhibitors of protein synthesis also extended the half-life of the gag precursor polypeptide, Pr76, suggesting that a host factor(s) may be required for the efficient intracellular processing of this polypeptide to the gag proteins.

Overexpression of Mos, Ras, Src, and Fos inhibits mouse mammary epithelial cell differentiation.

Mammary epithelial cells terminally differentiate in response to lactogenic hormones. We present evidence that oncoprotein overexpression is incompatible with this hormone-inducible differentiation and results in striking cellular morphological changes. In mammary epithelial cells in culture, lactogenic hormones (glucocorticoid and prolactin) activated a transfected beta-casein promoter and endogenous beta-casein gene expression. This response to lactogenic hormone treatment was paralleled by a decrease in cellular AP-1 DNA-binding activity. Expression of the mos, ras, or src (but not myc) oncogene blocked the activation of the beta-casein promoter induced by the lactogenic hormones and was associated with the maintenance of high levels of AP-1. Mos expression also increased c-fos and c-jun mRNA levels. Overexpression of Fos and Jun from transiently transfected constructs resulted in a functional inhibition of the glucocorticoid receptor in these mouse mammary epithelial cells. This finding clearly suggests that glucocorticoid receptor inhibition arising from oncogene expression will contribute to the block in hormonally induced mammary epithelial cell differentiation. Expression of Src resulted in the loss of the normal organization and morphological phenotype of mammary epithelial cells in the epithelial/fibroblastic line IM-2. Activation of a conditional c-fos/estrogen receptor gene encoding an estrogen-dependent Fos/estrogen receptor fusion protein also morphologically transformed mammary epithelial cells and inhibited initiation of mammary epithelial differentiation-associated expression of the beta-casein and WDNM 1 genes. In response to estrogen treatment, the cells displayed a high level of AP-1 DNA-binding activity. Our results demonstrate that high cellular AP-1 levels contribute to blocking the ability of mammary epithelial cells in culture to respond to lactogenic hormones. This and other studies indicate that the oncogene products Mos, Ras, and Src exert their effects, at least in part, by stimulating cellular Fos and probably cellular Jun activity.

Changes in phosphatidylinositol metabolism correlated to growth state of normal and Rous sarcoma virus-transformed Japanese quail cells.

Second-passage Japanese quail embryo cell cultures, normal or quantitatively transformed by Rous sarcoma virus, were investigated for phospholipid composition and metabolism. Cells cultivated at low and high population density as well as in the presence or absence of serum, have been compared by chemical analysis and in pulse-chase experiments. No differences in the lipid compositions between the normal and the tumor cells or between cells under different culture conditions were detected. In no case was the metabolism of phosphatidylserine or sphingomyelin affected by culture conditions. The metabolism of the choline and ethanolamine glycerophospholipids, however, differed according to culture conditions, whether cells were normal or transformed. Significantly, in normal cells, the breakdown of [32P]phosphate-labeled phosphatidylinositol was slowed when cell growth was restricted, i.e., at high population density or in medium without serum. This effect was not observed in tumor cells under such culture conditions, and cells were not growth inhibited. Hence, release of [32P]phosphate from phosphatidylinositol is the only parameter in the metabolism of phospholipids observed to correlate with growth.

Megakaryocytic differentiation of K562 cells is associated with changes in the cytoskeletal organization and the pattern of chromatographically distinct forms of phosphotyrosyl-specific protein phosphatases.

We have analyzed morphological and biochemical changes occurring during megakaryocytic differentiation of the human chronic myelogenous leukemia cell line K562 induced by phorbol 12-myristate 13-acetate (PMA). PMA-treated cells became growth arrested, were slightly larger and irregular in shape, adhered better to the culture flask surface, and expressed the glycoprotein IIIa on their surfaces. The morphological changes induced by PMA treatment were associated with the disappearance of actin from the cytosol and presumably reflect PMA-induced actin polymerization. Megakaryocytic differentiation was accompanied by about a 3-fold decrease in the specific phosphotyrosine protein phosphatase (PTPase) activity in the particulate membrane fraction, whereas the activity in the soluble cytosol fraction increased about 3-fold. The decrease of PTPase activity in the particulate membrane fraction could be attributed to the disappearance of at least 1 distinct PTPase form displaying an apparent native Mr of 200,000 and a reduction in activity of a Mr 43,000 PTPase found associated with membranes of all cells examined to date. The increase of PTPase activity in the cytosol fraction manifested itself by the appearance of a new Mr 40,000 PTPase and a reduction of a Mr 60,000 PTPase. These results suggest the existence of several growth- and/or differentiation-related PTPase activities in K562 cells.

The transformation of primary and established mouse mammary epithelial cells by p21-ras is concentration dependent.

We constructed a retroviral vector (pZSR) which is capable of simultaneously expressing the neomycin resistance gene and the viral ras oncogene. Primary mammary gland epithelial cells were prepared from mid-pregnant mice and infected with this virus. Cell lines with epithelial cell characteristics could be established with a low frequency. High expression of p21 v-ras was observed in these cells. They are tumorigenic and form soft agar colonies dependent on the presence of EGF and insulin in the growth medium but progress to hormone independent growth at higher passage numbers. A cloned cell line of non-tumorigenic, established mammary epithelial cells (NOG8) was also infected with the v-ras expressing virus. Individual cell clones expressing increasing amounts of p21 v-ras were selected. The level of p21 v-ras expression directly influences the morphology of the epithelial cells in culture, determines their cloning efficiency in soft agar and their tumorigenicity.

Purification and characterization of particles containing RNA-dependent DNA polymerase, in the allantoic fluid of uninfected leukosis virus-free chicken eggs.

The allantoic fluid of embryonated chicken eggs regularly contains particle-associated RNA-dependent DNA polymerase, as shown by its reaction with homopolymeric and heteropolymeric RNA and by the characterization of the products. The purification of the particles is described. The purified particles are different from the known avian RNA tumor viruses in their protein composition and their sedimentation constant. They do not exhibit biological properties typical for RNA tumour viruses, such as helper activity, interfering properties or infectivity and do not show endogenous DNA synthesis. The particles are discussed as non-viral elements.

Apoptosis-linked in vivo regulation of the tissue transglutaminase gene promoter.

Tissue transglutaminase (tTG) is upregulated in various cells undergoing apoptosis. To investigate the transcriptional regulation of tTG a mouse strain carrying a beta-galactosidase reporter gene under the control of a 3.8 kilobase fragment of the tTG promoter was characterised. The transgene construct was shown to be expressed in the apoptotic regions of the mouse embryo. Here we report that the regulation of the transgene is also apoptosis-linked in adult animals. The transgene is induced in endocrine apoptosis involving mammary gland involution and corpus luteum regression. Induction of the reporter gene is detectable during in vivo but not in vitro apoptosis of thymocytes induced by the glucocorticoid receptor, the nur77, p53 and the retinoid receptor gamma mediated pathways. Additionally, the lacZ expression mimics the activation of the endogenous promoter in tissues characterised by high apoptotic turnover. These results suggest that the apoptosis-specific transcriptional regulation of tTG is mediated through elements of a 3.8 kb promoter and may require cosignals available only in tissue environment. Cell Death and Differentiation (2000) 7, 1225 - 1233.

Apoptosis in the rat mammary gland and ventral prostate: detection of cell death-associated genes using a coincident-expression cloning approach.

Apoptosis plays a striking role in the hormone-dependent involution of the mammary gland, but it has proved difficult to distinguish between the 'cell death' associated genes and the 'tissue remodelling' genes which are expressed concurrently. To identify cell death-associated genes, we have established a 'coincidence analysis' based on the previously described 'RNA differential display' method of Liang and Pardee (1992). Coincidence analysis allows the detection of genes expressed during related processes in different organs and was employed here to identify transcripts in which expression patterns are seen to be associated with apoptosis during involution of both rat mammary- and the ventral prostate glands. That the coincidence analysis is a promising approach can be seen from the fact that while widely accepted apoptosis markers such as transglutaminase (Fesus et al, 1987; Strange et al, 1992) and sulfated glycoprotein-2 (Buttyan et al, 1989; Strange et al, 1992; Guenette et al 1994) exhibited similar expression in both regressing tissues, transcription of tissue remodelling enzymes was minimal in the involuting prostate. We describe here the characteristics of five clones isolated which show coincident expression during programmed cell death in mammary and prostate tissues. Partial sequence analysis revealed for three clones high homologies with previously described genes; the putative rat homolog of the growth arrest gene gas-1 (Schneider et al, 1988; Del Sal et al, 1992), an homolog of the mouse 'Integrin Associated Protein' (IAP) (Brown et al, 1990; Lindberg et al, 1993) and the sequence encoding for the 'Allograft Inflammatory Factor' AIF-1 (Autieri et al, 1995; Utans et al, 1995). One clone displayed homology with an expressed human sequence tag and one clone unrelated to any known DNA sequence was isolated. The expression of these genes in involuting rat mammary and ventral prostate, was correlated with that in other organs and in situ hybridization was applied to establish that the secretory epithelial cells which undergo programmed cell death are the site of elevated expression during the course of involution. Furthermore, we conclude that the coincidence analysis approach described here could be easily applied to facilitate the characterization of gene expression i.e. for the detection and comparison of hormonally regulated genes in different organs.

Isolation of cell death-associated cDNAs from involuting mouse mammary epithelium.

Although apoptosis is important in determining cell fate and maintaining tissue homeostasis, the initiation and control of apoptotic cell death in epithelium is not well understood. Post-lactationai involution of the mammary gland provides both an important developmental process and a normal physiological setting for studying apoptosis of epithelium. We used a differential screening strategy, based on previous studies correlating morphology with gene expression and nucleic acid integrity during mammary gland involution, to isolate genes involved in the regulation and execution of apoptotic cell death in regressing mammary epithelium. This screening strategy yielded a large number of genes the expression of which is significantly altered during mammary gland involution. These include genes associated with cell death processes, tissue remodelling and mesenchymal differentiation. In addition, a number of novel genes have been isolated. We have used Northern analysis and in situ hybridisation to study the expression of a selection of these putative death-associated genes during post-lactational mouse mammary gland involution.

Physiological apoptosis in hormone-dependent tissues: involvement of caspases.

Physiological apoptosis in mammals is a type of programmed cell death, an important element in the developmental repertoire ensuring tissue homeostasis and proper disposal of cells that are no longer needed, such as milk-producing epithelial cells in the mammary gland after lactation, luteal cells in the post partum Corpus luteum or secretory cells in the prostate after castration. Although incompletely described, apoptosis in hormone-dependent tissues is apparently initiated and executed using common biochemical strategies. These include survival pathways governed by local and systemic factors and hormones, diverse regulatory pathways and caspase-dependent execution pathways. Using an antibody that recognizes processed effector caspases or a fluorogenic caspase substrate, we present for the first time evidence that caspases are activated in the mammary gland, in the prostate and in the ovary at the time when apoptosis occurs. Most likely phagocytosis of apoptotic cells by neighboring cells may represent an important step, since only a modest involvement of professional phagocytes is apparent. Here, we will summarize and discuss recent data and will attempt to draw a generalized picture of how physiological apoptosis may occur in these organs.

Rous sarcoma virus p19 and gp35 can be chemically crosslinked to high molecular weight complexes. An insight into virus assembly.

We have used the method of chemical crosslinking in order to determine the spatial interactions between components of Rous sarcoma virus. A high molecular weight complex formed by crosslinking has been isolated by ultracentrifugation on sucrose density gradients containing 0.1% (w/v) sodium dodecyl sulphate. This complex is composed of the two viral glycoproteins gp85 and gp35, the gag protein p19, and the viral RNA. Two types of bonding are important for the formation and stability of the complex: first, native disulphide bonds between gp85 and gp35 and between individual p19 molecules; and second, hetero-crosslinking between gp35 and p19 as well as homo-crosslinking between p19. Although viral RNA is quantitatively present in the complex, experiments with RNase treatment show that it is not essential for its formation or stability. A small amount of lipid is present in the complex and appears to be crosslinked to p19. In vitro-labelling of purified virus with the lipophilic photoactivatable reagent [125I] iodonaphthylazide resulted in the labelling of gp35 and p19/23. In vivo-labelling of virus with [3H]palmitate resulted in only gp35 becoming labelled. These results substantiate the membrane association of these proteins. The significance of the interactions in the high molecular weight complex for the stability of the virus and, by implication, the role which they may play in viral assembly are discussed.

Expression and activation of tissue transglutaminase in apoptotic cells of involuting rodent mammary tissue.

Apoptosis is a form of cell death in which cellular integrity is maintained and neither cytoplasmic nor nuclear content is released. The Ca(2+)-dependent tissue transglutaminase (tTG) is an enzyme that forms protein cross-links between specific glutamyl and lysyl side-chains of intra- and extracellular proteins, therefore it may be responsible for the structural stabilization observed during the death process. In this study, the expression of tTG was investigated following the physiological process of forced weaning which results in an almost complete elimination of secretory epithelium by apoptosis and remodelling of the tissue structure. A dramatic induction of tTG was detected by immunoblotting of total mammary gland homogenates prepared from the involuting glands. The concentration of epsilon(gamma-glutamyl)-lysine crosslinks was also elevated in these samples, showing that the enzyme is activated during apoptosis. To determine the distribution of tTG and its relationship to apoptotic cells, paraffin-embedded specimens were studied by RNA in situ hybridization and immunohistochemical methods as well as using in situ labeling for nuclear fragmentation. All of these approaches indicated that mammary secretory epithelium expressed tissue transglutaminase coincident with the onset of apoptosis. The apoptotic and tTG-expressing cells were found to be identical as demonstrated by a histological double-labeling technique.

pp60c-src is a substrate for phosphorylation when cells are stimulated to enter cycle.

The endogenous cellular oncogene products, pp60c-src, exhibits a protein kinase activity, but is itself a phosphoprotein. Based on the assumption that pp60c-src might play a role in the control of cell proliferation, we have studied its behaviour as a substrate for phosphorylation known to occur when quiescent, serum-deprived cells are stimulated to enter cell cycle following addition of either serum, platelet-derived growth factor or the phorbol ester derivative, 12-O-tetradecanoyl-phorbol-13-acetate. For this purpose a partial purification of pp60c-src on DEAE ion-exchange chromatography was combined with immune precipitation. A 2-4-fold increase in serine phosphorylation of pp60c-src was consistently observed after stimulation of quiescent cells to growth.

Energy metabolism in the involuting mammary gland.

A strong and coordinated upregulation of the glycolytic, glutaminolytic and pentose phosphate pathway enzymes occurs during the onset of lactation in the normal mouse mammary gland. Induction of apoptosis by removing the pups led to an inactivation of the same enzymes with different time courses. While the ATP-consuming glycolytic 6-phosphofructo 1-kinase and mitochondrial bound hexokinase still remained high on days one and two of involution, the ATP-regenerating pyruvate kinase was immediately reduced. The enzymes of the pentose phosphate and glutaminolytic pathway were inactivated on the first two days of involution. In accordance with such an inactivation of the enzymes ATP, GTP, UTP, ADP, NAD NADH and lactate concentrations decreased. The synthetic product of UTP, UDP-N-acetylglucosamine, increased. AMP was found in the milk, not in the epithelial cells. The inactivation of the enzymes was caused by partial proteolysis or by a loss of the intact proteins from the cytosol without signs of proteolysis.

Oxygenation of tumors derived from ras transformed cells.

In order to gain insight into mechanisms governing the development of tumor hypoxia, malignancies derived from spontaneously tumorigenic or ras-transformed cell lines were grown in nude mice. As a rule, tumors with ras oncogenes exhibited rapid growth rates and large areas with low pO2 readings even at small tumor sizes. The slow proliferation rate of a spontaneously tumorigenic cell line was consistent with more adequate tissue oxygen levels. In all lines, hypoxia was accentuated at larger tumor sizes. These results demonstrate that ras transformation can lead to accelerated proliferation rates and is then concomitant with the development of pronounced tumor hypoxia.