Biomedical subjects

C Tomasetto

Publications and source records attributed to C Tomasetto.

50 records · Page 3Linked to original sources

PubMed1993

Specificity of gap junction communication among human mammary cells and connexin transfectants in culture.

In a previous paper (Lee et al., 1992), it was shown that normal human mammary epithelial cells (NMEC) express two connexin genes, Cx26 and Cx43, whereas neither gene is transcribed in a series of mammary tumor cell lines (TMEC). In this paper it is shown that normal human mammary fibroblasts (NMF) communicate and express Cx43 mRNA and protein. Transfection of either Cx26 or Cx43 genes into a tumor line, 21MT-2, induced the expression of the corresponding mRNAs and proteins as well as communication via gap junctions (GJs), although immunofluorescence demonstrated that the majority of Cx26 and Cx43 proteins present in transfected TMEC was largely cytoplasmic. Immunoblotting demonstrated that NMEC, NMF, and transfected TMEC each displayed a unique pattern of posttranslationally modified forms of Cx43 protein. The role of different connexins in regulating gap junction intercellular communication (GJIC) was examined using a novel two-dye method to assess homologous and heterologous communication quantitatively. The recipient cell population was prestained with a permanent non-toxic lipophilic dye that binds to membranes irreversibly (PKH26, Zynaxis); and the donor population is treated with a GJ-permeable dye Calcein, a derivative of fluorescein diacetate (Molecular Probes). After mixing the two cell populations under conditions promoting GJ formation, cells were analyzed by flow cytometry to determine the percentage of cells containing both dyes. It is shown here that Cx26 and Cx43 transfectants display strong homologous communication, as do NMEC and NMF. Furthermore, NMEC mixed with NMF communicate efficiently, Cx26 transfectants communicate with NMEC but not with NMF, and Cx43 transfectants communicate with NMF. Communication between Cx26 TMEC transfectants and NMEC was asymetrical with preferential movement of calcein from TMEC to NMEC. Despite the presence of Cx43 as well as Cx26 encoded proteins in the GJs of NMEC, few Cx43 transfectants communicated with NMEC. No heterologous GJIC was observed between Cx26- and Cx43-transfected TMEC suggesting that heterotypic GJs do not form or that Cx26/Cx43 channels do not permit dye transfer.

Blotting, Western↗

PubMed1993

The mouse one P-domain (pS2) and two P-domain (mSP) genes exhibit distinct patterns of expression.

We have previously shown that the human pS2 gene, which codes for a secreted peptide of 60 amino acids, is expressed in a number of human carcinomas, including carcinomas of the breast, the pancreas, and the large bowel. Strong pS2 gene expression was also observed in the normal gastric mucosa and in the regenerative tissues surrounding ulcerous lesions of the gastrointestinal tract. A number of pS2 similar peptides, designated as P-domain peptides, have been described, notably the porcine (PSP), murine (mSP), and human (hSP) spasmolytic polypeptides, which correspond to duplicated pS2 proteins. We have now cloned a mouse homolog of the human pS2 cDNA to dispose of an animal model to study the pS2 protein function, which remains unknown at the present time. We show that the mouse putative pS2 protein sequence and the physiological pattern of expression of the mouse pS2 gene are well conserved. The mouse pS2 gene is highly expressed in the stomach mucosa cells, whereas no pS2 gene expression could be detected in the mouse mammary gland, even during postnatal development processes dependent on growth factors or hormones. Using in situ hybridization, we show that although coexpressed in the fundus, the antrum and the antrum-pyloric regions of the stomach, the mouse pS2 and mSP genes exhibit distinct and complementary cellular patterns of expression.

Aging↗

PubMed1992

Down-regulation of a member of the S100 gene family in mammary carcinoma cells and reexpression by azadeoxycytidine treatment.

A cDNA clone, designated CaN19 (originally called clone 19), isolated by subtractive hybridization, contains sequences that are preferentially expressed in normal mammary epithelial cells but not in breast tumor cells. Comparison of its deduced amino acid sequence with sequences in the GenBank data base revealed similarity with the S100 protein family, a group of small Ca(2+)-binding modulator proteins involved in cell cycle progression and cell differentiation. CaN19 expression is down-regulated in normal cells by A23187, a calcium ionophore, suggesting that its regulation is calcium-dependent. We have assigned CaN19 to human chromosome 1q21-q24, a region containing four other S100-related genes. In contrast to CaN19 mRNA expression, most members of the S100 protein family are activated or overexpressed in tumor cells. Synchronization experiments by growth-factor deprivation demonstrated a biphasic induction of CaN19 expression in normal cells, approximately 2-fold in early G1 phase and another 2- to 3-fold at the G1/S boundary. Exposure of mammary tumor cells to 5-aza-2'-deoxycytidine, an inhibitor of DNA methylation, reactivated the expression of CaN19 mRNA.

Amino Acid Sequence↗

PubMed1992

The gene encoding the human spasmolytic protein (SML1/hSP) is in 21q 22.3, physically linked to the homologous breast cancer marker gene BCEI/pS2.

The human spasmolytic protein, SML1/hSP, an inhibitor of spasmolytic activity and gastric acid secretion in the pig, has been shown to exhibit homology to the pS2 protein, an estrogen-dependent breast cancer marker. Moreover, SML1/hSP and pS2 are expressed at the same localization in the normal stomach and during healing of the gastrointestinal tract. Here we report the chromosomal localization, obtained by in situ hybridization, of the hSP gene (SML1) to chromosome 21 at 21q22.3. Using pulsed-field gel electrophoresis, we found SML1 to be within 230 kb of the BCEI/pS2 gene.

Biomarkers, Tumor↗

PubMed1992

Transcriptional downregulation of gap-junction proteins blocks junctional communication in human mammary tumor cell lines.

Subtractive hybridization, selecting for mRNAs expressed in normal human mammary epithelial cells (NMECs) but not in mammary tumor cell lines (TMECs), led to the cloning of the human gap junction gene connexin 26 (Cx26), identified by its sequence similarity to the rat gene. Two Cx26 transcripts derived from a single gene are expressed in NMECs but neither is expressed in a series of TMECs. Northern analysis using rat Cx probes showed that Cx43 mRNA is also expressed in the normal cells, but not in the tumor lines examined. Connexin genes Cx31.1, Cx32, Cx33, Cx37, and Cx40 are not expressed in either normal cells or the tumor lines examined. In cell-cell communication studies, the normal cells transferred Lucifer yellow, while tumor cells failed to show dye transfer. Both Cx26 and Cx43 proteins were immunolocalized to membrane sites in normal cells but were not found in tumor cells. Further analysis demonstrated that Cx26 is a cell-cycle regulated gene expressed at a moderate level during G1 and S, and strongly up-regulated in late S and G2, as shown with lovastatin-synchronized NMECs. Cx43, on the contrary is constitutively expressed at a uniform low level throughout the cell cycle. Treatment of normal and tumor cells with a series of drugs: 5dB-cAMP, retinoic acid, okadaic acid, estradiol, or TGFb had no connexin-inducing effect in tumor cells. However, PMA induced re-expression of the two Cx26 transcripts but not of Cx43 in several TMECs. Thus Cx26 and Cx43 are both downregulated in tumor cells but respond differentially to some signals. Modulation of gap-junctional activity by drug therapy may have useful clinical applications in cancer.

Amino Acid Sequence↗

PubMed1992

Association of the human spasmolytic polypeptide and an estrogen-induced breast cancer protein (pS2) with human pancreatic carcinoma.

The human pS2 gene, isolated from the breast carcinoma cell line MCF-7 and shown to be under estrogen transcriptional control in a subclass of breast cancer cells was reported to be secreted in normal stomach surface epithelial cells, whereas additional gastrointestinal tissues like pancreas and colon do not secrete pS2 at all. In porcine pancreas, a spasmolytic polypeptide (sharing domains of homology with pS2) was observed; a corresponding human gene (hSP) was shown to be active in normal stomach mucosa. hSP and pS2 gene activity in normal and neoplastic pancreas tissues was then compared. Whereas both genes are inactive in normal pancreatic cells, activation of the pS2 sequence in a primary pancreatic carcinoma cell culture and in 23 tumor tissues was noted when investigated by immunostaining. In all cases when pS2 showed a regular 0.6 kb transcript, hSP displayed a transcript of 0.7 kb. Six of these tumors showed a reduced pS2 immunoreactivity and, at the same time, aberrant pS2 mRNA bands and a complete shut-down of the hSP gene were noted. In one case, whereas normal pancreas remained negative, the corresponding tumor and its metastasis displayed regular transcripts of pS2 and hSP. This remarkably high correlation suggests that pS2 and hSP expression in the pancreatic tumors, but not in their corresponding healthy tissue is significantly linked to molecular steps leading to tumorigenesis.

Adenocarcinoma↗

PubMed1991

Positive selection of candidate tumor-suppressor genes by subtractive hybridization.

A positive selection system designed to identify and recover candidate tumor-suppressor genes is described. The system compares mRNA expression of genes from normal and tumor-derived human mammary epithelial cells grown in a special medium that supports similar growth rates of the two cell types. mRNAs uniquely expressed in normal cells are recovered as cDNAs after subtraction with mRNA from tumor cells. Seven different clones, from 0.6 to 4.8 kilobases in transcript size and including both rare and abundunt transcripts, were recovered in the first 23 clones analyzed. Among the isolated clones were genes encoding the gap-junction protein connexin 26, two different keratins, and glutathione-S-transferase pi, as well as an unknown gene in the S100 family of small calcium-binding proteins. In principle, tumor-suppressor genes include two classes: class I, in which loss of function results from mutation or deletion of DNA and class II, in which loss of function is from a regulatory block to expression. A class II suppressor gene is assumed to be regulated by a different suppressor gene that lost its function by mutation or deletion. Both classes of tumor-suppressor genes may provide valuable proteins with clinical applications in cancer diagnosis or therapy. Class II suppressors may be especially useful because the normal genes are present and their reexpression may be inducible by drugs or other treatments.

Breast↗

PubMed1991

Induction of pS2 and hSP genes as markers of mucosal ulceration of the digestive tract.

The recently discovered pS2 protein is expressed under estrogen control in a subset of estrogen receptor-positive breast cancers and in an estrogen-independent manner in normal stomach mucosa. The pS2 gene belongs to a family of genes encoding peptides that contain a conserved 5-cysteine domain, the P domains. Although the function of the pS2 protein is unknown, it has been suggested that it may have cell growth stimulatory activity. We report here that expression of the pS2 gene in the digestive tract, which is normally restricted to the stomach, is strongly induced by mucosal ulcerations elsewhere in the tract, most notably in Crohn's disease. pS2 gene expression is restricted to the mucosal layers adjacent to the ulcerations, in a region where a novel epidermal growth factor-secreting cell lineage was shown to be induced by mucosal ulceration. The human hSP gene, which contains a tandem duplication of the pS2 gene P domain and is coexpressed with the pS2 gene in normal stomach mucosa but not in breast cancers, is also expressed in Crohn's disease. We suggest that pS2 gene expression may provide a useful marker for mucosal ulcerations of the digestive tract.

Biomarkers↗

PubMed1991

Vaccinia recombinants expressing secreted and transmembrane forms of breast cancer-associated epithelial tumour antigen (ETA).

Monoclonal antibody H23 identifies a polymorphic epithelial tumour antigen (ETA) that is aberrantly expressed in breast cancer and which may afford a target for active immunotherapy. We recently reported the cloning of H23-ETA genomic and cDNA clones. H23-ETA contains a multiple internal tandem repetition of a 20 amino acid motif and sequence analysis predicted two mRNA species encoding different ETA proteins, one harbouring a C-terminal potentially transmembrane hydrophobic zone (T) and a second form (S) that lacks this zone. We report that both RNA species can be detected in breast cancer cells. To further characterize the encoded proteins we have constructed vaccinia virus recombinants, VV-ETA-S and VV-ETA-T, separately expressing the alternative forms. Despite selective loss of internal tandem repeat elements during propagation of recombinant vaccinia, the encoded polypeptides were efficiently recognized by H23 monoclonal antibody. Immunoprecipitation revealed that ETA encoded by the S recombinant was secreted into the culture medium whereas the T form remained tethered at the cell surface. Both forms were readily detected in infected cells by immunofluorescence. Abnormal mobility of the T polypeptide indicated post-translational cleavage that may permit the extracellular domain of the T-polypeptide to be shed from the cell surface. Further, fluorescence-activated cell sorting analysis shows that the S form of the polypeptide is also partly present at the cell surface. Vaccinia recombinants expressing ETA may be of utility in the active immunotherapy of breast cancer.

Antigens, Neoplasm↗

PubMed1990

Vaccination against polyoma virus (PyV) tumors using vaccinia-PyV recombinants: a major tumor-specific transplantation antigen (TSTA) epitope resides within the C-terminal segment of middle-T protein.

We previously reported that inoculation of rats with live vaccinia virus (VV) recombinants VVpyMT, VVpyLT expressing either the middle-T (MT) or large-T (LT) proteins of polyomavirus (PyV) can elicit immunity to challenge with syngeneic PyV-tumor cells. We now report the results of cross-vaccination studies. VVpyMT was ineffective against cells expressing LT protein but prevented development of MT-expressing cells. Conversely, the VVpyLT was ineffective against MT-expressing cells. In the two experiments performed, tumor growth enhancement rather than retardation was observed in VVpyLT-vaccinated animals receiving PyV-LT (FRLTI) challenge tumor cells. To determine the location of the major TSTA within MT, a further VV recombinant (VVpyMT/Cfr) was constructed that expresses only the unique C-terminal segment of MT. VVpyMT-Cfr and VVpyMT were equally effective in eliciting tumor immunity, indicating the presence of a major TSTA epitope within the unique C-terminal region of MT.

Amino Acid Sequence↗

PubMed1990

hSP, the domain-duplicated homolog of pS2 protein, is co-expressed with pS2 in stomach but not in breast carcinoma.

Approximately 50% of human breast tumors secrete a small cysteine-rich protein, pS2, of unknown function. pS2 protein was recently found to be homologous to a porcine protein with hormonogastric activity, pancreatic spasmolytic polypeptide (PSP), in which the 5-cysteine domain present in pS2 is tandemly duplicated. We have characterized cDNA species encoding PSP and its human and mouse counterparts, hSP and mSP. We show that hSP and pS2 are separately encoded in the genome, and that the two proteins are co-expressed in normal stomach epithelium. However, expression of hSP was not detected in breast tumors. Computer analysis revealed that the pattern of conserved cysteine residues in hSP and pS2, the P domain, is present at the N termini of two other mammalian proteins, intestinal sucrase-isomaltase and lysosomal alpha-glucosidase.

Amino Acid Sequence↗

PubMed1990

Epidermal growth factor (EGF/URO) induces expression of regulatory peptides in damaged human gastrointestinal tissues.

The pS2 gene encodes for a small cysteine-rich protein, and was originally found by differential screening of a cDNA library from the human breast carcinoma cell line, MCF-7. The presence of pS2 is closely correlated with oestrogen dependence in breast carcinomas. While the function of pS2 is unknown, pS2 protein has been shown to be homologous with the gastrointestinal peptide hormone pancreatic spasmolytic polypeptide (PSP) and its human counterpart hSP, in which a 5-cysteine domain is tandemly repeated. The 5' flanking region of the pS2 gene contains an enhancer region responsive to oestrogens and to epidermal growth factor (EGF/URO). We now report that pS2 and hSP expression occurs in a wide range of endodermally-derived tissues, including the duodenum, the pancreas, and in a recently defined cell lineage associated with chronic gastrointestinal ulceration. In each case, this expression was associated with secretion of immunoreactive EGF/URO. We further show that the co-expression of pS2 and hSP in gastric surface epithelial cells is also associated with the secretion of EGF/URO in the subjacent mucous neck cells. Our results indicate that local EGF/URO secretion induces pS2 and hSP in adjacent cells, and that these molecules are then available to participate in pathophysiological responses. The finding of similar patterns of EGF/URO, hSP and pS2 expression in association with chronic damage suggests that this is a fundamental response in the healing of these tissues.

Cell Line↗

PubMed1989

Breast cancer protein PS2 synthesis in mammary gland of transgenic mice and secretion into milk.

PS2, a small estrogen-inducible secretory polypeptide with structural analogies to a growth factor, is produced by approximately 50% of human breast tumors. The function of PS2 is, however, unknown. To determine whether PS2 may play an autocrine role in the development of mammary tumors we constructed transgenic mice bearing fusion constructs designed to direct the expression of human PS2 in the lactating mammary gland under the control of the whey acidic protein (WAP) promoter. Mouse lines bearing the genomic PS2 gene under the control of the WAP promoter region (WAP-PS2-2) failed to express the transgene. However, mice harboring the fusion construct WAP-PS2-1, in which the PS2 coding sequence is inserted into the 5' untranslated region of the complete WAP gene, were observed to express the transgene. Expression was restricted to the secretory epithelium of the mammary gland during lactation, and PS2 protein was secreted into the milk. Nevertheless, no mammary gland dysplasia was observed, and PS2 expression had no discernable effect upon the physiology and/or development of the suckling young or the transgenic mother.

Animals↗

PubMed1988

Breast cancer-associated pS2 protein: synthesis and secretion by normal stomach mucosa.

The human pS2 gene is specifically expressed under estrogen transcriptional control in a subclass of estrogen receptor-containing human breast cancer cells. The pS2 gene encodes an 84-amino acid protein that is secreted after signal peptide cleavage. The distribution of pS2 protein in normal human tissues was studied with antibodies to pS2; pS2 was specifically expressed and secreted by mucosa cells of the normal stomach antrum and body of both female and male individuals. Moreover, no estrogen receptor could be detected in these cells, indicating that pS2 gene expression is estrogen-independent in the stomach. The function of the pS2 protein in the gastrointestinal tract is unknown. However, the pS2 protein is similar in sequence to a porcine pancreatic protein that has been shown to inhibit gastrointestinal motility and gastric secretion.

Amino Acid Sequence↗