p53 inhibits alpha 6 beta 4 integrin survival signaling by promoting the caspase 3-dependent cleavage of AKT/PKB.

Although the interaction of matrix proteins with integrins is known to initiate signaling pathways that are essential for cell survival, a role for tumor suppressors in the regulation of these pathways has not been established. We demonstrate here that p53 can inhibit the survival function of integrins by inducing the caspase-dependent cleavage and inactivation of the serine/threonine kinase AKT/PKB. Specifically, we show that the alpha6beta4 integrin promotes the survival of p53-deficient carcinoma cells by activating AKT/PKB. In contrast, this integrin does not activate AKT/PKB in carcinoma cells that express wild-type p53 and it actually stimulates their apoptosis, in agreement with our previous findings (Bachelder, R.E., A. Marchetti, R. Falcioni, S. Soddu, and A.M. Mercurio. 1999. J. Biol. Chem. 274:20733-20737). Interestingly, we observed reduced levels of AKT/PKB protein after antibody clustering of alpha6beta4 in carcinoma cells that express wild-type p53. In contrast, alpha6beta4 clustering did not reduce the level of AKT/PKB in carcinoma cells that lack functional p53. The involvement of caspase 3 in AKT/PKB regulation was indicated by the ability of Z-DEVD-FMK, a caspase 3 inhibitor, to block the alpha6beta4-associated reduction in AKT/PKB levels in vivo, and by the ability of recombinant caspase 3 to promote the cleavage of AKT/PKB in vitro. In addition, the ability of alpha6beta4 to activate AKT/PKB could be restored in p53 wild-type carcinoma cells by inhibiting caspase 3 activity. These studies demonstrate that the p53 tumor suppressor can inhibit integrin-associated survival signaling pathways.

Integrin alpha 6/beta 4 complex is located in hemidesmosomes, suggesting a major role in epidermal cell-basement membrane adhesion.

The alpha 6/beta 4 complex is a member of the integrin family of adhesion receptors. It is found on a variety of epithelial cell types, but is most strongly expressed on stratified squamous epithelia. Fluorescent antibody staining of human epidermis suggests that the beta 4 subunit is strongly localized to the basal region showing a similar distribution to that of the 230-kD bullous pemphigoid antigen. The alpha 6 subunit is also strongly localized to the basal region but in addition is present over the entire surfaces of basal cells and some cells in the immediate suprabasal region. By contrast staining for beta 1, alpha 2, and alpha 3 subunits was very weak basally, but strong on all other surfaces of basal epidermal cells. These results suggest that different integrin complexes play differing roles in cell-cell and cell-matrix adhesion in the epidermis. Immunoelectron microscopy showed that the alpha 6/beta 4 complex at the basal epidermal surface is strongly localized to hemidesmosomes. This result provides the first well-characterized monoclonal antibody markers for hemidesmosomes and suggests that the alpha 6/beta 4 complex plays a major role in epidermal cell-basement membrane adhesion. We suggest that the cytoplasmic domains of these transmembrane glycoproteins may contribute to the structure of hemidesmosomal plaques. Immunoultrastructural localization of the BP antigen suggests that it may be involved in bridging between hemidesmosomal plaques and keratin intermediate filaments of the cytoskeleton.

Microdistribution of specific rat monoclonal antibodies to mouse tissues and human tumor xenografts.

Detailed evaluations of the microdistribution of 125I-labeled monoclonal antibodies (MoAbs) to normal tissue antigens were conducted in BALB/c mice. MoAb 273-34A, which binds to a target molecule on the lumenal surface of lung endothelial cells, localizes quickly and efficiently throughout the lung vasculature. MoAb 133-13A, which binds to an antigen on macrophage-like cells expressed in nearly equal amounts in lung, liver, and spleen, localizes most efficiently to spleen and less well to liver and lung. The microdistribution of MoAb 133-13A in liver and spleen is consistent with the antigen distribution in these organs, but in the lung a more diffuse microdistribution is observed, indicating poor access of MoAb to the antigen-positive alveolar macrophages. These findings are consistent with the hypothesis that tight endothelium (lung) represents a significant barrier to extravasation of MoAb into tissue while fenestrated (spleen) and sinusoidal (liver) endothelium are more easily penetrated. In human tumor bearing nu/nu mice, the microdistribution of MoAb to the beta 4 and alpha 6 subunits of integrin was studied. These MoAbs do not cross-react with murine integrins and thus are tumor-specific in the nu/nu mouse model. Localization of 125I-labeled MoAb 450-11A, which reacts with an intercellular domain of beta 4 integrin, is very weak and diffuse. All MoAbs to extracellular domains (mouse 450-9D, 450-30A1, and rat 439-9B) localize well to the tumor. Microdistribution of these MoAbs in the 3 different tumors is nonuniform with heavy distribution near the blood vessels, whereas antigen distribution as determined by immunoperoxidase shows a much more uniform pattern throughout the tumors. In experiments with 125I-labeled MoAb 439-9B F(ab')2, the nonuniform pattern of distribution was not changed. Gross and microdistribution of different doses of 125I-labeled MoAb 439-9B were studied. The percent of injected dose per g of MoAb in the tumor at 48 h did not vary significantly (P greater than 0.1) up to a dose of 500 micrograms/mouse, and active MoAb was recovered in comparable amounts in the serum from animals in all doses. In contrast, the microdistribution of MoAb at the high dose was different than that at low doses. At doses up to 100 micrograms/mouse, a perivascular pattern was obtained, whereas at 500 micrograms/mouse the 125I-labeled MoAb was distributed nearly evenly throughout the tumor. These data indicate that high doses of MoAb penetrate deeply into portions of the tumor that are distant from blood vessels.(ABSTRACT TRUNCATED AT 400 WORDS)

Monoclonal antibody to human carcinoma-associated protein complex: quantitation in normal and tumor tissue.

We have identified and quantitated a tumor protein complex, TSP-180, on murine carcinomas with two monoclonal antibodies (MoAbs) (Cancer Res., 46: 707-712, 1986). One of the two MoAbs, 135-13C, recognizes a TSP-180-like protein complex on several human carcinomas in culture. MoAb 135-13C has been used to purify the human TSP-180 complex from A431 cells and the purified material used to immunize F344 rats to produce another MoAb, 439-9B, to the human TSP-180 complex. This MoAb does not precipitate the murine TSP-180 or bind to murine cells. Both MoAb 135-13C and 439-9B precipitated the same proteins from A431 cells but did not compete with each other for binding sites, indicating that they recognize different epitopes on the same protein. The two MoAbs have been used in a two-site assay to quantitate TSP-180 proteins on human cells and tissues. Carcinoma cell lines A431, SW948, and A549 all give high values (46 to 443 ng/mg of protein) while murine tumors, a human melanoma, and human fibroblasts are negative (less than 10 ng/mg of protein). Most tissues from autopsy of 2 normal individuals are negative for human TSP-180 at the levels tested (less than 10 ng/mg of protein). Some organs have intermediate range expression: spleen, 5 to 111 ng/ml of protein; colon, 24 to 111; and small intestine, 39 to 99. One primary colon and one larynx tumor were positive (144 to 372 ng/mg of protein) while 5 breast carcinomas, a stomach tumor, a metastatic melanoma, and a kidney tumor were negative. These data indicate that human TSP-180 may be preferentially expressed in certain malignant carcinomas of diverse origin. The potential for TSP-180 as a tumor marker requires further study.

Expression of tumor antigen correlated with metastatic potential of Lewis lung carcinoma and B16 melanoma clones in mice.

Expression of a tumor-associated antigen, recognized by a monoclonal antibody (MoAb 135-13C) to lung carcinoma cells, has been studied in cloned Lewis lung carcinoma (3LL) and in B16 melanoma (F1 and F10) tumor lines endowed with different metastatic potentials. MoAb 135-13C recognizes a protein complex (tumor-specific Mr 180,000 protein) that appears on the cell surface of several murine lung carcinomas but is not detected on normal cells in culture. Standard metastatic variants of B16 melanoma (F1 and F10) and two variant sublines of 3LL (M1087 and BM21548) together with the parental line of 3LL have been used for these experiments. The two cloned variant lines derived from 3LL have been shown to retain high (M1087) and low (BM21548) metastatic phenotypes during in vivo passaging. We found that all three cell lines of 3LL bind monoclonal antibody specifically, but one cell variant with higher metastatic potential shows a higher capacity to bind MoAb 135-13C than did the other variant. Similarly we found that B16 F10 cells bind higher amounts of MoAb 135-13C than did B16 F1 cells. In addition the analysis of the amounts of MoAb 135-13C bound to the cell surface of several other in vitro and in vivo tumor lines with different metastatic capacity demonstrates that all tumor lines which express high ability to colonize to the lung also express, on the cell surface, higher amounts of tumor-specific Mr 180,000 protein. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiograms of immunoprecipitates from cell lysates of 3LL and B16 tumor lines demonstrate that MoAb 135-13C specifically precipitated three proteins banding at molecular weights of 204,000, 134,000, and 116,000. We conclude that MoAb 135-13C recognizes a surface protein complex which is present in higher amounts in 3LL and B16 cells which possess higher capacity to metastasize to the lung.

Ligand-induced phosphorylation of a murine tumor surface protein (TSP-180) associated with metastatic phenotype.

A tumor surface protein (TSP-180) has been identified on murine lung carcinomas using two monoclonal antibodies (MoAbs) (135-13C and 346-11A). Quantitative analysis of TSP-180 on 3LL variants maintained either in vitro or in vivo indicates that TSP-180 is highly expressed in highly malignant metastatic cells. In reducing conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis banding patterns of TSP-180 obtained with MoAb 135-13C from cell lysates of 3LL metastatic cells show three proteins migrating to Mr 204,000, 134,000, and 116,000. In the same experimental conditions MoAb 135-13C precipitates from low metastasizing ones only one band, corresponding to the lower molecular weight (Mr 116,000). All bands of TSP-180 observed in 3LL variants are labeled by lactoperoxidase-catalyzed radioiodination of viable cells, incorporate 32PO4, and contain carbohydrates, as judged by binding to wheat germ agglutinin. These results indicate that all proteins have external exposure on the cell surface and that at least some of TSP-180 proteins could be differentially regulated in different tumor cells (highly metastatic versus low metastatic). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis banding patterns and immunoblots obtained from cell lysates of 3LL variants by using a monoclonal antibody to phosphotyrosine (IG-2) indicate that this MoAb recognizes proteins migrating with molecular weights identical to those reported for TSP-180. Moreover, the immunoblots of solubilized immunocomplex, obtained from cell lysates of 3LL variants by using MoAb 135-13C, demonstrate that MoAb IG-2 specifically reacts with TSP-180 proteins. Experiments undertaken in order to assess if some or all of TSP-180 proteins have tyrosine kinase activity demonstrate that MoAb 135-13C binding to the cell surface induces specific phosphorylation of the Mr 204,000 protein of TSP-180. Phosphoaminoacid analysis of the ligand-induced phosphorylated protein (pp204) demonstrates that this protein is phosphorylated at serine and tyrosine. Results reported lead us to hypothesize that TSP-180 is involved in growth-regulation mechanisms and that its high expression on cells with more malignant phenotype could be responsible for a proliferative advantage of such tumor clones.

Integrin (alpha 6/beta 4) expression in human lung cancer as monitored by specific monoclonal antibodies.

In this study, the expression of the alpha 6/beta 4 integrin complex was analyzed in human lung carcinomas both in vitro and in vivo, using two monoclonal antibodies which recognize the integrin subunits alpha 6 (Mab 135-13C) and beta 4 (Mab 439-9B). Immunoprecipitation patterns obtained from established human lung carcinoma cell lines demonstrated that the alpha 6 and the beta 4 subunits were differentially expressed in carcinomas of different types. The alpha 6 subunit was expressed in all the cell lines tested (squamous cell carcinoma A431, adenocarcinoma A549, large cell carcinoma DG3, and small cell carcinoma AE2). The beta 4 subunit was expressed in non-small cell cancer lines but was not detectable in the small cell cancer line tested. Using a quantitative two-site assay, we measured the concentration of the alpha 6/beta 4 integrin in matched biopsies from primary lung tumors and from normal lung. These studies confirmed that the complex was differentially expressed in non-small versus small cell lung cancers and that it was also detectable in lysates from normal lung at low levels. The highest levels of alpha 6/beta 4 were found in moderately differentiated squamous cell carcinomas. By immunohistochemistry, the beta 4 subunit was detectable in all the squamous cell carcinoma and adenocarcinomas tested (a total of 59), but not in 10 small cell cancers. The patterns of immunoreactivity were consistent with the expected distribution of membrane glycoproteins and, in some squamous cell carcinomas, were suggestive of the localization displayed by molecules involved in carcinoma-stroma interaction. Immunohistochemical staining indicated that beta 4 was also expressed in specific types of nonrespiratory pulmonary epithelial cells.

Stimulation of tumor cell growth in vitro by a monoclonal antibody to a tumor specific protein (TSP-180) present on the cell surface of 3LL cells.

Proliferation capacity and MHC class I antigen expression of two Lewis lung carcinoma (3LL) metastatic variants (C87, BC215) grown under defined experimental conditions (serum-free defined medium or 10 per cent serum) have been studied following exposure to MoAb 135-13C which recognizes on these cells a tumor surface protein of 180,000 daltons (TSP-180). The results of this study indicate that the high metastatic clone (C87) binds higher amounts of MoAb to TSP-180 and Db antigens than does the low metastatic one (BC215), while both clones express very low amounts of Kb antigens. 3LL clones grown in 10 per cent serum or adapted in serum-free, defined medium show the same metastatic phenotype and MHC class I antigen expression, but when grown in defined medium exhibit increased capacity to bind MoAb 135-13C. However, the relative binding rate of 3LL clones grown in 10 per cent serum or in defined medium is unchanged: the high metastatic clone always showing higher capacity to bind MoAb to TSP-180. Furthermore, comparison of EGF binding sites on the cell surface of 3LL clones, grown in different culture conditions, demonstrates that the C87 clone binds higher amounts of labelled EGF and that this amount increases in serum-free defined medium, exactly as reported for TSP-180. In addition, competition experiments demonstrated that MoAb 135-13C does not compete for EGF binding sites on 3LL cell surface. Studies on cell proliferation following exposure to MoAb 135-13C, revealed that the low metastatic clone (BC215) is more actively stimulated than the high metastatic one. Moreover, similar data were obtained after exposure of 3LL clones to physiological amounts of different growth factors (i.e. EGF, MSA, insulin). Analysis of MHC class I antigen expression following exposure to MoAb 135-13C indicated that MoAb 135-13C induces on the cell surface of the C87 clone a transient low modulation of Db antigens. These results suggest that 3LL cells endowed with lower metastatic potential are more dependent on the microenvironmental conditions than the high metastasizing ones, and that MoAb 135-13C binding to 3LL cell surface stimulates proliferation as reported for several known growth factors.

Retinoic acid negatively regulates beta 4 integrin expression and suppresses the malignant phenotype in a Lewis lung carcinoma cell line.

Retinoic acid (RA) is a potent inhibitor of the malignant phenotype and of tumour cell growth. We observed that in vitro RA treatment of a highly metastatic lung carcinoma cell line (C87) induced a marked reduction in the amount of the beta 4 integrin subunit. The downregulation of this adhesion molecule was assessed by immunofluorescence, immunoprecipitation, and northern analysis. In order to investigate the effects of RA on the malignant phenotype in C87 cells we performed morphological and functional analysis after RA treatment. We found that RA was able to produce marked changes in C87 cell shape, increasing the number of flat cells (90% of the total cell population), and significantly inhibiting the malignant and invasive phenotype of C87 cells. RA treatment suppressed their clonogenic potential in soft agar (control, 20 +/- 5; RA, 0), and strongly reduced their chemotactic and chemoinvasive capacity (chemotaxis: control, 231 +/- 5; RA, 28 +/- 0; chemoinvasion: control, 132 +/- 11; RA = 2 +/- 1). FACS analysis and cell count, however, indicated that RA reduced the growth of C87 cells only partially. After 72 h of treatment we observed only a 10% reduction in the S phase fraction of the cell population. Finally, the reduced lung colony-forming ability, observed after i.v. injection of RA-treated cells (lung foci/animal: RA-treated cells, 1 +/- 0.1; untreated, 8.5 +/- 0.8), further supports the conclusion that in this murine lung carcinoma cell line a marked reduction in the expression of the beta 4 integrin subunit is associated with a marked inhibition of the malignant phenotype.

Growth-inhibition of ht29 cells exposed to N-methylformamide correlates with altered expression of alpha-6/beta-4 integrin.

The anticancer agent N-methylformamide (NMF) suppresses the expression of the c-myc proto-oncogene in human colon carcinoma cells while increasing the doubling time and reducing tumorigenicity in these cells. However, the mechanism by which NMF exerts its effects has remained unclear. We compared the expression of c-myc and of other growth-regulated genes (p53, beta-actin) to that of the H3 histone gene, which is specifically expressed in S-phase cells, in HT29 human colon carcinoma cells maintained in vitro or in nude mice. The growth fraction of the cell populations in the presence or absence of NMF was also evaluated at different days of growth by flow cytometric analysis. To assess whether prolonged exposure to NMF might induce a different phenotype in human carcinoma cells, the expression of alpha6, beta1, and beta4 integrin subunits were evaluated. The data indicate that NMF treatment induces a reduction in the growth fraction of HT29 colon carcinoma cells accompanied by a reduction in c-myc, H3 histone, p53, and beta-actin gene expression, and that prolonged exposure to NMF induces elevated expresssion of alpha6/beta4 integrin receptor, These data suggest that NMF-induced reduction of the proliferative capacity supports a different 'maturation status' of colon carcinoma cells which is defined by an elevated expression of the alpha6/beta4 integrin.

Integrin Beta-4 expression in colorectal-cancer.

Integrin alpha 6 beta 4 plays an important role in the interaction of epithelia with basement membranes, and its expression appears to be profoundly altered during tumor progression. Using a quantitative immunochemical assay, we investigated the expression of the beta 4 subunit associated with alpha 6 in 25 primary carcinomas, and in matching normal mucosae. alpha 6 beta 4 was expressed in all the carcinoma and mucosa samples. The highest beta 4 levels were detected in tumors at high clinical stage (Dukes' stage C). Furthermore, beta 4 reactivity inversely correlated with the degree of differentiation. By immunohistochemistry,beta 4 expression was particularly strong in the epithelium lining the upper third of the crypts and the absorbing surface of normal mucosa. In villous adenomas, beta 4 immunostaining tended to be enhanced in the epithelium lining the outer surfaces of neoplastic villi, but only 5 of 8 samples tested scored positive. In carcinomas, beta 4 expression was detected in 18 of 21 samples tested, and was strongly influenced by the pattern of tumor growth and by the type and level of differentiation. Carcinomas, or areas of carcinomas, with cohesive and differentiated growth pattern demonstrated weak beta 4 expression at the tumor-stroma interface. Carcinoma cells at the lumenal surface of the intestine, and carcinomas, or areas of carcinomas, composed of small clusters of cells surrounded by stroma, demonstrated strong beta 4 expression. Altogether, our observations indicate that in colorectal tumors the expression of the beta 4 subunit is strongly influenced by microenvironmental factors and tends to increase in high stage, poorly differentiated lesions.

Integrin b4 expression in peripheral-nerve tumors.

The alpha 6 beta 4 integrin complex is expressed in epithelial, endothelial and nerve cells. We analyzed the immunohistochemical expression of the beta 4 subunit in normal peripheral nerves, in neurofibromas associated with type 1 neurofibromatosis and in sporadic neurofibrosarcomas. In normal peripheral nerves (4 samples), the beta 4 integrin was diffusely expressed at the level of the perinevrium and at the interface between axons and Schwann cells. In neurofibromas (6 cases), beta 4 was undetectable or markedly decreased relative to normal peripheral nerves. Neurofibrosarcomas (3 cases) were immunohistochemically negative for beta 4 expression. These observations suggest that a down-regulation of the alpha 6 beta 4 integrin is associated with the neoplastic progression of peripheral nerve tumors.

Metastatic phenotype: growth factor dependence and integrin expression.

Homogeneous subpopulations, which are endowed with low or high metastatic potential, were selected from Lewis lung carcinoma (3LL) in an attempt to correlate metastatic phenotype with specific properties of tumor cells. Since the growth of malignant cells at secondary sites could depend on their ability to respond to microenvironments, the growth factor dependence of 3LL variants has been studied. The ability of variant lines to grow in monolayer and in soft agar cultures, either in the presence or absence of different growth factors or serum, was analyzed and correlated with their metastatic potential. The reported results demonstrate that tumor cells expressing higher metastatic potential also exhibit higher capacity to grow and proliferate in all the culture conditions tested, independently of the addition of exogenous growth factors or serum. Moreover, since highly metastasizing cells express a significant amount of TGF-beta 1 mRNA, a pattern of autocrine growth is postulated for 3LL metastatic cells. One relevant aspect of the phenotype of transformed cells is their reduced adhesion to solid substrates; this phenomenon is thought to reflect the invasive and metastatic potential of tumor cells. Since the adhesion of the cells to substrata is mediated by molecules of the extracellular matrix, the expression of extracellular matrix receptors (integrins) was studied on 3LL metastatic variants. In particular, through immunochemical and biochemical studies we investigated the expression of the laminin receptor(alpha 6/beta 1) and of a novel receptor (integrin: alpha 6/beta 4), of unknown function. The receptors were quantitated on the cell surface of 3LL variants by the use of specific monoclonal antibodies which recognize, respectively, different epitopes of alpha 6, beta 4 or beta 1 subunits. Results demonstrate that the novel integrin alpha 6/beta 4, is specifically expressed in highly metastasizing 3LL cells, whereas the laminin receptor alpha 6/beta 1 is expressed in all 3LL variants. In conclusion, data presented demonstrate that 3LL cells endowed with higher metastatic potential are more independent of the microenvironmental conditions in that they possess a higher autocrine capacity than the lower metastasizing ones, and could acquire higher capacity to invade through the expression on their cell surface of specific receptors for cell adhesion (the novel integrin, defined as alpha 6/beta 4).

MHC antigens expressed on 3LL metastatic variants: correlation with the expression of a TSP-180 protein.

In attempts to correlate metastatic potential with specific properties of tumor cells, homogeneous subpopulations, which are endowed with low or high metastatic potential, have been selected from Lewis lung carcinoma (3LL). In particular, since cell surface constituents are possibly involved in the metastatic process, changes in antigen expression have been correlated with the metastatic potential of 3LL variants. In this view, we quantitated the expression of MHC (Db,Kb) antigens and of a tumor specific protein (TSP) identified by the monoclonal antibody (MoAb) 135-13C on some "in vitro" and "in vivo" variants of 3LL. The MoAb 135-13C was found to recognize a TSP-180 protein that appears on the cell surface of several murine carcinomas, but is not detected on normal cells in culture. Studies of the MHC expression on these variants, by the use of the indirect immunofluorescent staining or the direct binding of the MoAb to H-2Db (28-14-8) and the MoAb to H-2Kb (28-13-3), demonstrate that "in vivo" and "in vitro" 3LL variants which, are endowed with a higher metastatic potential, express on the cell surface a higher amount of the Db antigen. By contrast, all the 3LL lines have few cells recognized by the MoAb to H-2Kb and express low amounts of this antigen on the cell surface. The direct binding to different tumor lines and the analysis of the immunoprecipitates from the cell lysates by the use of the MoAb 135-13C demonstrate that the TSP-180 protein is highly expressed on 3LL cells which possess high capacity to metastasize to the lung. The variations induced in 3LL metastatic phenotype by the injection of the variant lines in allogeneic mice (Balb/c, C3HeB:H-2d,H-2k, respectively) or after treatment with the specific MoAb 135-13C have, also, been studied. An attempt was made to correlate the changes in 3LL metastatic phenotype with the expression of the TSP-180 protein and of the MHC antigens. We conclude that a high expression on the cell surface of the Db antigen and of the TSP-180 protein, is associated with a high malignant phenotype of 3LL tumor cells.

Modulation of heat shock protein synthesis in two human melanoma cell lines.

Two human melanoma cell lines, largely different from one another in their intrinsic thermosensitivity, were exposed to supranormal temperatures and labeled with 35S-methionine. The protein patterns resolved by SDS-polyacrylamide gel electrophoresis showed in both cell lines an increased synthesis of a unique set of heat shock proteins (HSP) of 72 Kdalton (KD). Already evident after 15 min at 42 degrees C, the relative rate of synthesis of these HSP increased progressively for up to 3 h of continuous heat treatment. The cells exposed for 1 h at 42 degrees C and then returned to 37 degrees C maintained a high relative rate of HSP synthesis for more than 6 h. The rate of decay of the neosynthesized HSP did not differ from that of the overall cell proteins. Since in both cell lines all the parameters concerning HSP induction were identical, no correlation can be established between their intrinsic sensitivity towards the conditioning treatment and the capacity to respond to heat treatment with an increased synthesis of these proteins.

Second generation monoclonal antibodies to the human integrin alpha 6 beta 4.

Second generation monoclonal antibodies to the alpha 6 beta 4 subunits of human integrins have been prepared. MAbs 450-9D, 10D, and 11A1 react at different sites on the beta 4 molecule and MAbs 450-30A1 and 33D react at the same site on the alpha 6 subunit. Double determinant (two-site) radioimmunoassays using combinations of these MAbs have been developed. Two assays for beta 4 distinguish between the whole beta 4 molecule and the beta 4 molecule truncated from the C-terminus (form c) while another assay measures the presence of alpha 6 subunits. Data from the two-site assays support the following conclusions: (1) Colon tumors and normal colon mucosa express large amounts of alpha 6 beta 4 although only form c of the beta 4 was detected; (2) There is no evidence for alpha 6 beta 1 expression in colon; however, some of this complex may be present in certain lung tumors. The extracellular domains of alpha 6 and beta 4 can associate with each other even if the cytoplasmic domain of the beta 4 subunit is not present. MAbs to specific domains of the beta 4 molecule may be useful in analyses of forms a and c in normal and malignant tissue. The fact that only the largest beta 4 molecule "a" retains the phosphorylation site may have functional significance.

[Psychopathology in nunneries]. / Zur Psychopathologie in Frauenklöstern.

The psychiatric observations of female adepts and nuns of Christian monastic congregations, seen as in- or outpatients of a mental hospital of an order, are reported, mainly focussing on schizophrenic and schizophreniform psychoses and severe personality disorders. The patient statistics and sketches of psychopathology and the dynamics of the disorders are presented. The selection process for adepts for the monastic profession and the need for education of the responsible leaders in psychological, psychotherapeutic and psychiatric issues are considered.

p53 is required for brown adipogenic differentiation and has a protective role against diet-induced obesity.

Proper regulation of white and brown adipogenic differentiation is important for maintaining an organism's metabolic profile in a homeostatic state. The recent observations showing that the p53 tumor suppressor plays a role in metabolism raise the question of whether it is involved in the regulation of white and brown adipocyte differentiation. By using several in vitro models, representing various stages of white adipocyte differentiation, we found that p53 exerts a suppressive effect on white adipocyte differentiation in both mouse and human cells. Moreover, our in vivo analysis indicated that p53 is implicated in protection against diet-induced obesity. In striking contrast, our data shows that p53 exerts a positive regulatory effect on brown adipocyte differentiation. Abrogation of p53 function in skeletal muscle committed cells reduced their capacity to differentiate into brown adipocytes and histological analysis of brown adipose tissue revealed an impaired morphology in both embryonic and adult p53-null mice. Thus, depending on the specific adipogenic differentiation program, p53 may exert a positive or a negative effect. This cell type dependent regulation reflects an additional modality of p53 in maintaining a homeostatic state, not only in the cell, but also in the organism at large.

Inhibition of p85, the non-catalytic subunit of phosphatidylinositol 3-kinase, exerts potent antitumor activity in human breast cancer cells.

The phosphoinositide 3-kinases (PI3Ks) are heterodimers consisting of the catalytic subunit p110 and the regulatory subunit p85. The PI3K/Akt pathway is strongly deregulated in breast cancer (BC) representing one of the mechanisms of resistance to therapies. Therefore, the identification of inhibitors of PI3K components represents one of the main goals to produce therapeutic agents. Here, we evaluated the efficacy of a phosphopeptide 1257 (P-1257) that targeting p85 strongly inhibits PI3K activity. We tested the effects of P-1257 administration in vitro and in vivo using BC cells expressing different levels of ErbB-2 and resistant or responsive to Trastuzumab. We demonstrated that inhibition of p85 activity by P-1257 induces cell death and sensitizes JIMT-1 and KPL-4 ErbB-2-overexpressing BC cells to Trastuzumab treatment. It is noteworthy that P-1257 delivery in vivo by electroporation or liposomes significantly inhibits the proliferation of tumor cells engrafted at subcutaneous and visceral sites. Overall, our data indicate that the p85 subunit is a valid target for therapeutic approaches and suggest that the structure of the peptide used in our study could be utilized for the development of novel drugs to apply in combination with therapies that fail to cure BCs with high PI3K activity.

Analysis of translation products synthesized in isolated rat hepatocytes treated with diethylnitrosamine.

Isolated rat hepatocytes were labeled with 35S-methionine in the presence of 25 mM diethylnitrosamine (DENA). The intrinsically labeled proteins were analyzed by one- and two-dimensional gel electrophoresis and the fluorographic patterns were compared with those obtained from untreated hepatocytes. The results of short term experiments (2 h) show that, in the presence of 25 mM DENA, protein synthesis is inhibited by 50%. This reduction encompasses all protein species without selective inhibition of certain proteins.