Chromosomal locations of the murine T-cell receptor alpha-chain gene and the T-cell gamma gene.

Two independent methods were used to identify the mouse chromosomes on which are located two families of immunoglobulin (Ig)-like genes that are rearranged and expressed in T lymphocytes. The genes coding for the alpha subunit of T-cell receptors are on chromosome 14 and the gamma genes, whose function is yet to be determined, are on chromosome 13. Since genes for the T-cell receptor beta chain were previously shown to be on mouse chromosome 6, all three of the Ig-like multigene families expressed and rearranged in T cells are located on different chromosomes, just as are the B-cell multigene families for the Ig heavy chain, and the Ig kappa and lambda light chains. The findings do not support earlier contentions that genes for T-cell receptors are linked to the Ig heavy chain locus (mouse chromosome 12) or to the major histocompatibility complex (mouse chromosome 17).

Tumor progression in four mammary epithelial cell lines derived from the same patient.

Two primary and two metastatic cell lines with distinct phenotypes and genotypes have been established from a patient diagnosed as having infiltrating and intraductal mammary carcinoma (21T series). All four lines can be cultured in the same medium, DFCI-1, which also supports long-term growth of normal epithelial cells. Therefore, we have been able to compare normal and tumor cells at the cellular and molecular levels. The mammary origin of the 21T series was confirmed by using antibodies against the human milk fat globule antigen-2 epitope. The two primary tumor lines (21NT and 21PT) are both immortal and aneuploid, although only 21NT is tumorigenic in the nude mouse assay. The two populations derived from the metastatic pleural effusion (21MT-1 and 21MT-2) each exhibit distinct characteristics in morphology and growth factor requirements. The erbB2 gene is amplified and overexpressed in all of these cell lines compared to normal epithelial cell controls. These four tumor cell lines from a single patient represent a mammary tumor progression series that has been established in long-term cell culture.

Insulin-like growth factor (IGF)-binding protein-related protein-1: an autocrine/paracrine factor that inhibits skeletal myoblast differentiation but permits proliferation in response to IGF.

Skeletal myogenic cells respond to the insulin-like growth factors (IGF-I and IGF-II) by differentiating or proliferating, which are mutually exclusive pathways. What determines which of these responses to IGF skeletal myoblast undergo is unclear. IGF-binding protein-related protein 1 (IGFBP-rP1) is a secreted protein with close homology to the IGF-binding proteins (IGFBPs) in the N-terminal region. IGFBP-rP1, previously called mac25 and IGFBP-7, is highly expressed in C2 skeletal myoblasts during the proliferative phase, but is down-regulated during myoblast differentiation. To determine the role of IGFBP-rP1 in myogenesis, IGFBP-rP1 was overexpressed in C2 myoblasts using a retroviral vector. Western blots indicated that the resulting C2-rP1 myoblasts secreted approximately 27-fold higher levels of IGFBP-rP1 than control C2-LX myoblasts that were transduced with a control vector (LXSN). Compared with C2-LX myoblasts, the differentiation responses of C2-rP1 myoblasts to IGF-I, IGF-II, insulin, and des(1-3)IGF-I were significantly reduced (P < 0.05). However, proliferation responses of C2-rP1 and C2-LX myoblasts to these same factors were not significantly different. Exposure of control C2-LX myoblasts to factors secreted by C2-rP1 myoblasts using a transwell coculture system reduced C2-LX myoblast differentiation significantly (P < 0.05). Experiments with the mitogen-activated protein kinase (MAPK) kinase inhibitor PD098059 suggested that IGFBP-rP1 inhibits a MAPK-dependent differentiation pathway. In confirmation of this idea, levels of phosphorylated extracellular signal-regulated kinase-2 (a MAPK) were reduced in C2-rP1 myoblasts compared with those in C2-LX myoblasts. These findings indicate that IGFBP-rP1 may function as an autocrine/paracrine factor that specifies the proliferative response to the IGFs in myogenesis.

SEMP1, a senescence-associated cDNA isolated from human mammary epithelial cells, is a member of an epithelial membrane protein superfamily.

We have cloned a human cDNA, SEMP1 (senescence-associated epithelial membrane protein 1), using differential display (DD) of mRNA. We compared mRNA expression profiles between cultured normal senescent human mammary epithelial cells (HMECs) and proliferating, early passage HMECs. From the amino acid sequence of the open reading frame (ORF) of the cDNA, we infer that the protein belongs to a family of membrane-associated, epithelial cell-specific proteins. The translation product has 91% identity to a mouse protein, claudin-1, a tight junction (TJ)-associated protein. SEMP1 mRNA is expressed in human tissues, including adult and fetal liver, pancreas, placenta, adrenals, prostate and ovary but at low or undetectable levels in a number of human breast cancer cell lines. SEMP1 is a member of a superfamily of epithelial membrane proteins (EMPs), which may have multiple potential functions, including maintenance and regulation of cell polarity and permeability, perhaps through mechanisms involving tight junctions.

Age-related increase in methylation of ribosomal genes and inactivation of chromosome-specific rRNA gene clusters in mouse.

An age-related increase in DNA methylation of the multicopy 18S and 28S ribosomal RNA genes was found in CBA/Ca mice beginning between 6 and 18 months of age at the 5' end of these genes in liver, brain and spleen. The highest level of age-associated hypermethylation was mapped to the proximal 5' spacer domain. Silver staining of actively transcribing ribosomal genes in metaphase chromosomes from stimulated spleen cells provided cytological evidence that these mice have 3 rRNA cistrons located on chromosomes 15, 16, and 18. The ribosomal gene cluster located on chromosome 16 was preferentially inactivated in older animals. Exposure of spleen cells from older individuals to 5-azacytidine appeared to both reactivate ribosomal gene clusters and reduce rRNA gene methylation.

Expression and function of tight junction associated molecules in human breast tumor cells is not affected by the Ras-MEK1 pathway.

Constitutive activation of Ras or Ras-mediated signaling pathways is one of the initial steps during tumorigenesis that promotes neoplastic transformation. Recently it was reported that in Ha-Ras overexpressing MDCK cells the tight junction proteins claudin-1, occludin and ZO-1 were absent at cell-cell contact sites but present in the cytoplasm. Inhibition of MEK1 activity recruited all three proteins to the cell membrane leading to a restoration of the tight junction barrier function in MDCK cells. In order to evaluate the relevance of the MEK1 pathway in tight junction regulation in breast cancer cells, we investigated the effect ofMEK1 inhibition on expression of claudin-1, occludin and ZO-1 in natively claudin-1 expressing T47-D cells (low Ras activity), claudin-1 negative MCF-7 cells (elevated Ras activity) as well as two retroviral claudin-1 transduced MCF-7 daughter cell lines with prominent membrane and cytoplasmic claudin-1 dominant homing, respectively. Although we effectively blocked phosphorylation of MAPKs ERK-1 and ERK-2 using the selective MEK1 inhibitor PD98059, no quantitative changes of mRNA or protein levels of claudin-1, occludin and ZO-1 could be detected in all cell lines investigated. Furthermore, immnfluorescence analysis of claudin-1 revealed that inhibition of the MAPK pathway did not alter th e subcellular cytoplasmic distribution of claudin-1 to be more membrane specific. Finally, the diffusion barrier properties of tight junctions as analyzed by transepithelial resistance (TER) or paracellular flux analysis of 3 and 40 kDa dextran of tight junctions were not altered in the claudin-1 positive T47-D and the MCF-7 cell lines. Our findings indicate that the proposed involvement of the Ras-MEK-ERK pathway is likely not involved in the dysregulated tight junction formation in breast tumor cells and indicates that elevated activity of Ras might not be of general importance for the disruption of tight junction structures in breast tumors.

Preferential chromosome loss in human papilloma virus DNA-immortalized mammary epithelial cells.

Human papilloma virus (HPV) DNA-immortalized human mammary epithelial cells may provide a model system for studying the molecular basis of immortalization and its role in breast neoplasia. Cytogenetic analyses were performed on clones derived from HPV 16- and HPV 18-immortalized human mammary epithelial cells. The majority of the clones contained near-diploid karyotypes. The single most frequent whole-chromosome loss was that of chromosome 19. Regions that showed preference for deletion and/or translocation included 2pter, 11qter, and 15pter. Evidence of chromosome 19 loss was confirmed by polymerase chain reaction (PCR)-generated, chromosome 19-specific, dinucleotide microsatellite repeat polymorphism analysis.

Enhanced expression of an insulin growth factor-like binding protein (mac25) in senescent human mammary epithelial cells and induced expression with retinoic acid.

mac25, the subject of this report, was selected by the differential display of mRNA method in a search for genes overexpressed in senescent human mammary epithelial cells. mac25 had previously been cloned as a discrete gene, preferentially expressed in normal, leptomeningial cells compared with meningioma tumors. mac25 is another member of the insulin growth factor-binding protein (IGFBP) family. Insulin-like growth factors are potent mitogens for mammary epithelial cells, and the IGFBPs have been shown to modulate this mitogenic activity. We report here that mac25, unlike most IGFBPs, is down-regulated at the transcription level in mammary carcinoma cell lines, suggesting a tumor-suppressor role. The gene was mapped to chromosome 4q12. We found that mac25 accumulates in senescent cells and is up-regulated in normal, growing mammary epithelial cells by all-trans-retinoic acid or the synthetic retinoid fenretinide. These findings suggest that mac25 may be a downstream effector of retinoid chemoprevention in breast epithelial cells and that its tumor-suppressive role may involve a senescence pathway.

A partial methylation profile for a CpG site is stably maintained in mammalian tissues and cultured cell lines.

We wished to determine if a partial methylation profile for a specific CpG site was stably maintained in both mammalian tissues and cultured cell lines. To accomplish this, we identified a CpG site with a partial methylation profile located upstream of the mouse adenine phosphoribosyltransferase promoter region. This site was found to be methylated at a level of approximately 25% in mouse brain, kidney, lung, and skeletal muscle tissues, at a level close to 50% in liver, and at level close to 0% in testis. These tissue-specific methylation profiles were not altered during aging. A methylation profile of approximately 25% at this CpG site was also observed in five mouse teratocarcinoma stem cell lines and one additional cultured cell line. This profile, however, was altered upon cellular differentiation, adenine phosphoribosyltransferase hemizygosity, and a loss of adenine phosphoribosyltransferase activity in some of the cultured cell lines. We conclude that partial methylation of a specific CpG site can be stably maintained both in vivo and in vitro and that a mechanism exists for its maintenance. The functional significance of a partial methylation profile remains to be determined.

Developmental regulation of Mac25/insulin-like growth factor-binding protein-7 expression in skeletal myogenesis.

Mac25 is a newly discovered member of the insulin-like growth factor-binding protein (IGFBP) family, recently assigned the name IGFBP-7, Mac25/IGFBP-7 is hypothesized to have growth-suppressing activity, since mac25/IGFBP-7 mRNA is down-regulated in several tumor cell lines and is highly expressed in senescent mammary epithelial cells. In this study, mac25/ IGFBP-7 mRNA expression was characterized in the C2 skeletal myogenic cell line, which undergoes a transition from actively dividing, undifferentiated myoblasts to nondividing, differentiated myotubes. Mac25/ IGFBP-7 mRNA levels were 2.5-fold higher in dividing C2 myoblasts than in nondividing myotubes. The inverse correlation between mac25/IGFBP-7 expression and myogenic differentiation was further examined by treating myogenic cultures with transforming growth factor-beta (TGF-beta) or insulin-like growth factor-I (IGF-I). TGF-beta inhibited myogenic differentiation by 98% and stimulated mac25/IGFBP-7 mRNA expression 2-fold. IGF-I stimulated differentiation by 50% and inhibited mac25/IGFBP-7 expression 2- to 3-fold. These findings indicate that, in contrast to other cell systems examined so far, expression of this new member of the IGFBP family is not always correlated with a nonproliferative state.

X-inactivation patterns in lymphocytes and skin fibroblasts of three cases of X-autosome translocations with abnormal phenotypes.

X-inactivation patterns were studied by replication analyses both in lymphocytes and skin fibroblasts of two patients carrying balanced X-autosome translocations, t(X;10)-(pter;q11) and t(X;17)(q11;q11), and one patient with an unbalanced translocation t(X;22)(p21;q11). Preferential late replication of the normal X chromosome was found in lymphocytes of both patients carrying balanced translocations and in skin fibroblasts of the patient carrying the translocation t(X;17). However, skin fibroblasts of the patient with a translocation t(X;10) showed preferential late replication of the abnormal der(X) chromosome with no spreading of late replication to the autosomal segment. In the case of unbalanced translocation t(X;22) there was preferential late replication of the der(X) chromosome both in lymphocytes and skin fibroblasts. The abnormal phenotype of the patients is discussed in relation to the observed X-inactivation patterns and the variability of the patterns in different tissues.

Antenatal diagnosis of mosaic trisomy 8 confirmed in fetal tissues.

An aneuploid karyotype with an extra submetacentric C-group chromosome was observed in all metaphase cells in 5 of 24 primary amniotic fluid cell clones and in admixture with normal cells in two additional clones. Trisomy 8 was demonstrated by R-banding. The parents elected to terminate the pregnancy. Successful cultures were initiated from nine fetal tissues and aneuploid cells were observed in cultures derived from two separate sites in the skin.

Down-regulation of retinoic acid receptor beta in mammary carcinoma cell lines and its up-regulation in senescing normal mammary epithelial cells.

Retinoids are important cellular, dietary factors that regulate differentiation and cellular growth. They serve as ligands for specific nuclear receptors, the retinoic acid receptors (RARs). Ligand-activated receptors regulate gene transcription through target retinoic acid-responsive elements (RAREs) found in promoter regions. We have investigated the expression of retinoic acid receptor genes (alpha, beta, gamma) and retinoid X receptor beta in normal, senescing, and tumorigenic human mammary epithelial cells. We find that most tumor cells show a loss of RAR-beta expression, but that RAR-alpha and -gamma as well as retinoid X receptor beta are variably expressed in both normal and tumor cells. RAR-beta gene expression is induced both by retinoic acid and by fenretinide in normal cells, but tumor cells fail to respond to either. In contrast, RAR-beta expression increases with serial passage in senescing cells. Paradoxically, both normal and tumor cells can trans-activate an exogenous beta-RARE, as demonstrated by reporter gene assays. Oligonucleotide mobility shift assays with the beta-RARE show a single discrete complex in normal cells, whereas tumor cells exhibit a heterogeneous set of larger complexes, which indicates that tumor cells utilize a different array of factors within the beta-RARE. Reporter gene assays with extended promoter regions indicate the presence of negative regulatory elements and/or factor binding sites that reside between -1500 and the RARE located at -59, and that the promoter is down-regulated in MCF-7 tumor cells. Our findings reveal a dichotomy: RAR-beta transcription is down-regulated in tumor cells compared with normal human mammary epithelial cells, and up-regulated in senescence.

Elevated retinoic acid receptor beta(4) protein in human breast tumor cells with nuclear and cytoplasmic localization.

The transcription factor retinoic acid receptor beta(2) (RARbeta(2)) is a potent inhibitor of breast cancer cells in vitro, and studies suggest that RARbeta expression is lost in primary breast cancer. Although RARbeta(2) is selectively down-regulated at the mRNA level in breast tumor cells, we show that expression of an RARbeta protein is elevated in five of five breast tumor cell lines relative to normal human mammary epithelial cells. Subsequent analysis identified this protein as the translation product of the human RARbeta(4) transcript. Unlike the previously characterized mouse RARbeta(4) isoform, the human RARbeta(4) retains only half of a DNA-binding domain and lacks a ligand-independent transactivation domain at its N terminus. The RARbeta(4) protein localizes to the cytoplasm and to subnuclear compartments that resemble nuclear bodies. The structure and preliminary characterizations of human RARbeta(4), coupled with the observation that its expression is greatly elevated in breast tumor cell lines, support the hypothesis that RARbeta(4) functions as a dominant-negative repressor of RAR-mediated growth suppression.

Genomic organization of claudin-1 and its assessment in hereditary and sporadic breast cancer.

Human claudin-1 is an integral protein component of tight junctions, a structure controlling cell-to-cell adhesion and, consequently, regulating paracellular and transcellular transport of solutes across human epithelia and endothelia. Recently, a claudin-1 (CLDN1) cDNA has been isolated from human mammary epithelial cells (HMECs). CLDN1 expression in HMECs, in contrast to low or undetectable levels of expression in a number of breast tumors and breast cancer cell lines, points to CLDN1 as a possible tumor-suppressor gene. In order to evaluate the CLDN-1 gene in sporadic and hereditary breast cancer, we have characterized its genomic organization and have screened the four coding exons for somatic mutations in 96 sporadic breast carcinomas and for germline mutations in 93 breast cancer patients with a strong family history of breast cancer. In addition, we have compared the 5'-upstream sequences of the human and murine CLDN1 genes to identify putative promoter sequences and have examined both the promoter and coding regions of the human gene in the breast cancer cell lines showing decreased CLDN1 expression. In the sporadic tumors and hereditary breast cancer patients, we have found no evidence to support the involvement of aberrant CLDN1 in breast tumorigenesis. Likewise, in the breast cancer cell lines, no genetic alterations in the promoter or coding sequences have been identified that would explain the loss of CLDN1 expression. Other regulatory or epigenetic factors may be involved in the down-regulation of this gene during breast cancer development.

9-cis-retinoids: biosynthesis of 9-cis-retinoic acid.

Retinoids function through conformational alterations of ligand-dependent nuclear transcription factors, the retinoic acid receptors, and retinoid X receptors. 9-cis-Retinoic acid is a known biological ligand for retinoid X receptors, but its synthesis pathway in vivo is largely unknown. Recently, we identified a cis-retinol dehydrogenase (cRDH) that oxidizes 9-cis-retinol to 9-cis-retinal. Since both the expression of cRDH mRNA and its substrate are found in liver, we studied 9-cis-retinol metabolism and 9-cis-retinoic acid biosynthesis in two hepatic-derived cell types, Hep G2 hepatoma cells and HSC-T6 stellate cells. Both cell lines accumulate similar amounts of 9-cis-retinol provided in the medium. However, Hep G2 cells preferentially incorporate all-trans-retinol when equimolar concentrations of all-trans- and 9-cis-retinol were provided. In contrast, HSC-T6 cells did not exhibit a preference between all-trans- and 9-cis-retinol under the same conditions. Esterification of 9-cis-retinol occurred in both cell types, likely by acyl-CoA:retinol acyltransferase and lecithin:retinol acyltransferase. In vitro enzyme assays demonstrated that both cell types can hydrolyze 9-cis-retinyl esters via retinyl ester hydrolase(s). In Hep G2 cells, 9-cis-retinoic acid synthesis was strongly inhibited by high concentrations of 9-cis-retinol, which may explain the low levels of 9-cis-retinol in liver of mice. Cell homogenates of Hep G2 can convert all-trans-retinol to 9-cis-retinal, suggesting that the free form of all-trans-retinol may be used as a source for 9-cis-retinol and, thus, 9-cis-retinoic acid synthesis. Our studies provide the basis for identification of additional pathways for the generation of 9-cis-retinoic acid in specialized tissues.

Expression of retinoic acid receptor beta mediates retinoic acid-induced growth arrest and apoptosis in breast cancer cells.

The expression of the retinoic acid receptor beta (RAR beta) mRNA is absent or down-regulated in most human breast cancer cell lines. To investigate the role RAR beta may have in regulating the proliferation of breast cancer cells, we used retroviral vector-mediated gene transduction to introduce the human RAR beta gene into two RAR beta-negative breast tumor cell lines, MCF-7 and MDA-MB-231. RAR beta-transduced clones underwent growth inhibition associated with G1 arrest when treated with 1 microM all-trans-retinoic acid (RA). Moreover, the MCF7-RAR beta transduced clones also underwent apoptosis after 4 to 6 days of RA treatment. The RA-induced growth arrest in MDA231-RAR beta transduced cells is associated with c-myc mRNA down-regulation, whereas the RA-mediated apoptosis of MCF7-RAR beta transduced cells is not associated with c-myc down-regulation. These observations suggest a critical role for RAR beta in mediating growth arrest and apoptosis in breast cancer cells.

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.