cDNA cloning and genomic analysis of a new multigene family sharing common phylogenetic and expression profiles with the laminin receptor gene.

We have isolated a human laminin receptor (LR) cDNA which bears a different sequence in its 5' end with regard to the corresponding region in the regular LR mRNA. This different sequence hybridizes to a 1 kb mRNA. We have cloned a 740 bp cDNA for this transcript (cDNA 48-1). Search on sequence data bases revealed no sequence homology with known messengers or proteins. Using cDNA 48-1 in a simplified version of the protocol with which we had previously characterized the LR gene as a member of a retrogene family in mammals, we show in the present paper that the gene of this new transcript exhibits phylogenic, expression and amplification features that strickingly recall those of the LR gene.

c-myc gene amplification in selected node-negative breast cancer patients correlates with high rate of early relapse.

In breast cancers with histologically negative axillary nodes selected for high frequency of recurrence, the amplification of c-myc, erbB-2 and int-2 genes was found to concern, respectively 25% (16/65), 31% (25/81) and 14% (10/70) of tumours. Their relation with tumour progression expressed by relapse-free survival is reported. Using univariate analyses, c-myc amplified tumours showed significant association with early (30-month period after diagnosis) (P = 0.0013) and intermediate (50-month period after diagnosis) (P = 0.0398) risks of recurrence. In contrast, only a trend towards higher relapse was observed in erbB-2 amplified breast cancers with respect to later events (occurring over the first 30-month period). Multivariate analyses indicated that c-myc amplification is an independent prognostic factor stronger than oestrogen receptor status and tumour size to define a high risk subset in node-negative patients selected for high frequency of recurrence.

Genomic analysis of the 67-kDa laminin receptor in normal and pathological tissues: circumstantial evidence for retroposon features.

We have cloned two cDNAs for the human 67-kDa laminin receptor (LR). In the present report we show that these clones hybridize to many restriction fragments in Southern experiments in human. This particular pattern is accounted for by the presence of up to 16 and 21 copies of the laminin receptor gene per haploid genome in human and mouse, respectively. In contrast, a single gene copy is found in chicken. Chromosomal localization reveals four main loci: LAMRP1, laminin receptor pseudogene 1 (Chr 3); LAMRP2, laminin receptor pseudogene 2 (Chr 12); LAMRP3, laminin receptor pseudogene 3 (Chr 14); LAMRP4, laminin receptor pseudogene 4 (Chr X). Comparison of our experimental results to the known features of processed retropseudogenes enabled us to conclude that the LR gene belongs to a retroposon family in mammals.

Distinct effects of gonadotropin-releasing hormone analogs and 4-hydroxytamoxifen on pS2 mRNA expression with respect to cell proliferation in MCF-7 breast cancer cells.

Gonadotropin-releasing hormone (GnRH) analogs and antiestrogens display direct antiestrogenic effects on the proliferation of hormone-sensitive breast cancer cells. This study aimed to determine whether growth inhibition of 17 beta-estradiol-stimulated MCF-7 breast cancer cells by the agonist D-Trp6 GnRH, the GnRH antagonist BIM 21009C and 4-hydroxy-tamoxifen (OHT) respectively occurred through alterations of the estrogen receptor (ER)-mediated intracellular pathway. The pS2 mRNA expression is primarily dependent on activated ER in MCF-7 cells, and pS2 protein could act as a growth factor. Drug effect on pS2 mRNAs were qualitatively compared to those on the cell cycle. Unlike OHT, GnRH analogs did not suppress the 17 beta-estradiol-induced pS2 mRNA expression whilst the cell cycle was blocked. The pS2 mRNA expression was induced by D-Trp6 GnRH alone without effect on the cell cycle. The outcome of our study is double. Firstly, GnRH analogs are distinct from OHT as regards their effects on the ER-mediated intracellular pathway. Secondly, pS2 mRNA expression is not strictly related to MCF-7 cell proliferation, suggesting that pS2 protein has a function other than that of critical growth regulator.

Modulation of tPA, PAI-1 and PAI-2 antigen and mRNA levels by EGF in the A431 cell line.

It has been reported that EGF treatment enhances uPA but not tPA in the A431 epidermoid carcinoma cell line. To determine whether the absence of tPA modulation by EGF could be due to the action of inhibitors, we assayed tPA, PAI-1, PAI-2 and tPA/PAI-1 complexes by immunological assays and zymography in A431 serum-free medium. We found that, under conditions in which EGF had no effect on tPA activity, tPA antigen increased with a concomitant rise of tPA/PAI-1 complexes, indicating the action of an inhibitor. Both tPA antigen and tPA/PAI-1 complexes were modulated by EGF in a time and concentration dependent manner. tPA/PAI-1 complex levels were lower than tPA levels, suggesting the presence of other inhibitors. Immunological assays detected PAI-2 in addition to PAI-1 and showed a time and dose response to EGF. Modulation of tPA and the anti-activators by the growth factor was confirmed by identification of the corresponding transcripts with cDNA probes. We conclude that the net plasminogen activator activity in A431 cells is the result of a balance between activators and inhibitors.

Modulable Southern and Northern blot hybridization apparatus for routine screening of gene amplification and expression.

An apparatus for Northern and Southern blot hybridization is described. It allows from one to twenty-four blots to be processed at the same time, with different probes. All the pre-, post- and hybridization steps are performed without handling the filters and the experimenter is totally protected from beta radiations. The development of such modulable materials has become necessary since Southern and Northern techniques are becoming routine assays in hospitals, particularly in the field of oncology, in prognosis and for hereditary diseases, as an antenatal diagnosis procedure.

A paradigm for oncogene complementation in human breast cancer.

Multiple cellular oncogenes are amplified in malignant tumours, and it is possible to invoke gene dosage as a possible base for product activity. In vitro data have shown that two different oncogenes can cooperate in converting a normal cell into one that is tumorigenic. This suggested that multiple cooperative alterations might be involved in cancer progression. Breast cancers have a broad spectrum of clinical behaviours ranging from highly aggressive neoplasms to almost chronic diseases. Fifty months of clinical follow-up were studied in 143 patients with primary breast cancers. Uni- and multivariate analyses were performed in order to determine any synergistic effect of amplified c-myc, erbB-2 and int-2 genes on the disease-free state and overall survival. We showed that c-myc amplification was associated with early recurrence and shorter survival; in contrast, erbB-2 and int-2 extra copies resulted in later relapse events, especially in patients whose tumours showed a normal copy number of c-myc genes. This pointed out sequential activation of complex regulatory cascades within the cell. Such particular behaviour enabled us to categorize erbB-2 and int-2 oncogenes into a group showing delayed action, in contrast to c-myc involvement in rapid spread of the tumour. As expected, co-amplified c-myc and erbB-2 genes showed positive cooperation with respect to recurrence and shortening of overall survival. Finally, the harmful effects of amplified c-myc and erbB-2 oncogenes were dramatically increased in patient subgroups showing a normal copy number of the int-2 gene. Multivariate analysis was used to evaluate disease-free survival and to test for potential interactions of oncogene covariates. It pointed out multiple independent combinations which enabled us to define complementation groups with respect to clinical patient behaviour.

Correlation of erbB-2 gene amplification with low levels of estrogen and/or progesterone receptors in primary breast cancer: do erbB-2 products delineate hormone-independent tumors?

We analyzed erbB-2 gene amplification in 170 primary breast carcinomas. Thirty-one percent of tumors exhibited additional copies of erbB-2 gene. Chi-square analysis did not elicit any association between gene amplification and either menopausal or node status. A slight trend was observed with respect to the SBR grading. In contrast, significant correlation was associated with the age of patients and we found a strong relation with the intratumoral steroid receptor status. ErbB-2 amplification significantly occurs in tumors whose estrogen receptor and progesterone receptor were below the cut-off value or absent and tumors with dissociated estrogen receptor and progesterone receptor status were revealed as entities similar to both estrogen receptor and progesterone receptor negative tumors.

A new member of the immunoglobulin superfamily--CTLA-4.

The immunoglobulin superfamily is a group of proteins, each made of one or several domains sharing key structural features with either the variable (V) or the constant (C) immunoglobulin domains. It includes such functionally important members as the immunoglobulins themselves, major histocompatibility complex (MHC) class I and class II and T-cell receptor (TCR) molecules. Several members of this superfamily are expressed on lymphocytes where they are membrane-bound and capable of interactions with other members of the family, thus taking part in cell-cell recognition. In screening mouse cytolytic-T-cell-derived cDNA libraries, we came across cDNA clones defining a sequence, CTLA-4, which could encode a 223-amino-acid protein clearly belonging to the immunoglobulin superfamily. It consists of one V-like domain flanked by two hydrophobic regions, one of which has a structure suggestive of membrane anchoring. CTLA-4 is mainly expressed in activated lymphocytes and is coinduced with T-cell-mediated cytotoxicity in inducible models of this process. The mouse ctla-4 gene maps to band C of chromosome 1.

Genetic mapping of a human class II antigen beta-chain cDNA clone to the SB region of the HLA complex.

A class II antigen beta-chain cDNA clone was isolated from a human B-cell cDNA library by using as a probe the murine I-A beta gene. This cDNA clone, pHA beta, was shown to be distinct from the DC beta- and DR beta-related loci by DNA sequence analysis, thus suggesting that it might correspond to a third polymorphic human class II locus, SB, which encodes secondary B-cell antigens. Genetic mapping of this beta-chain cDNA clone to the SB region was performed by the blot hybridization procedure. We showed that (i) within panels of HLA-DR homozygous human B-cell lines and of unrelated individuals who have been typed for HLA antigens, differential mobility of DNA fragments segregated with distinct SB genotypes; (ii) gamma-ray-induced deletion mutants that have lost the expression of DR or DC/MT antigens but maintain SB expression preserved a pattern consistent with (a) their SB phenotype and (b) the genetic independence of the SB locus with respect to DR and DC/MT; and (iii) within an informative family, two siblings differing only for one allele at the SB locus (because of the occurrence of an internal recombination between DR and GLO) and otherwise HLA identical exhibited a restriction enzyme polymorphism linked to the SB locus. Therefore, all available data are compatible with identity between HA beta and SB beta.

Analysis of human class II antigen alpha chain genes: a summary.

A cDNA clone corresponding to the HLA-DR alpha chain has been isolated following immunoprecipitation of polysomes with a monoclonal antibody against the denatured DR alpha chain. This cDNA clone has been used to isolate and solve the complete structure of the DR alpha chain gene and a cDNA clone corresponding to the DC alpha chain. The DR alpha and the DC alpha chains bear remarkable homology to each other and have one domain (alpha 2) which is a member of the immunoglobulin superfamily. The DC alpha chain gene has been demonstrated to be the homologue of the murine I-A alpha chain gene, and it has been localized on chromosome 6 using deletion mutants. A restriction enzyme polymorphism of the DC alpha chain gene has been detected in HLA-DR homozygous typing cells. This report is a summary of a presentation made at the HLA-DR meeting held in Marseille, France.

A biochemical analysis of the secregation of HLA-DR heavy and light chains in a family studied by two-dimensional gel electrophoresis.

Previous studies in our laboratory using primed lymphocyte typing (PLT) in an informative family have led to the identification of four traits and two regions, separable by recombination within the HLA-DR system. In the present study, we analysed the heterogeneity of HLA-DR antigens among members of this family as a first step towards defining the molecular entities responsible for the observed cellular reactivities. Cell lines of various genotypes derived from the members of this family were labelled biosynthetically with 35S-methionine. The total glycoproteins and immunoprecipitates prepared with two monoclonal anti-HLA-DR antibodies, two monoclonal anti-mouse Ia antibodies cross-reactive with HLA-DR, and a rabbit anti-HLA-DR (anti-p27,33) antiserum were fractionated by two-dimensional gel electrophoresis. In agreement with previous results, HLA-DR light chains were clearly polymorphic whereas the heavy chains looked rather monomorphic. The light chain subunits either segregated with one haplotype, or were shared by some but not all haplotypes or were common to all haplotypes. Whereas the haplotype-specific polypeptides correlated best with HLA-DR3 and HLA-DR5 specificities, the shared subunits may have corresponded to the cross-reactivities observed by PLT. Comparison of the patterns obtained with the various monoclonal antibodies and with the rabbit antiserum revealed that each monoclonal antibody bound a subset of HLA-DR light chains, all of which were present in the anti-p27,33 precipitates. This rabbit antiserum and one of the monoclonal antibodies immunoprecipitated a set of heavy chains not bound by the other 3 antibodies. Since overlapping pools of light chains were present in all five immunoprecipitates, these results suggest that different heavy chains may associate with the same light chains.

Polymorphism and complexity of the human DC and murine I-A alpha chain genes.

A cDNA clone encoding the human B cell alloantigen DC alpha chain (pDCH1) has been used to analyse the structure of the human and murine major histocompatibility complexes by the DNA filter hybridization technique. The pDCH1 probe hybridizes to a single DNA sequence present on chromosome 17 in the mouse genome. A restriction enzyme polymorphism enables us to map this sequence to the I-A subregion. Extensive restriction enzyme polymorphism detected in HLA-DR homozygous typing cells is reminiscent of the DR/MT linkage disequilibrium groups, suggesting that the pDCH1 probe could be useful for haplotype typing in the human population. The HLA-DR region appears more complex than the I region since a second DC-like hybridizing sequence is detected in the human genome in these experiments.

cDNA clone for the heavy chain of the human B cell alloantigen DC1: strong sequence homology to the HLA-DR heavy chain.

A cDNA library has been constructed from a B cell mRNA fraction enriched for HLA-DR sequences, and cDNA clones corresponding to sequences specifically expressed in B lymphocytes have been isolated by a differential screening procedure. Analysis of these clones with probes specific for the HLA-DR heavy chain gene allowed the characterization of HLA-DR heavy chain-related sequences. One clone, pDCH1, was demonstrated to encode the DC1 heavy chain because the amino acid sequence predicted from its nucleotide sequence matches eight out of nine residues available for comparison in the amino-terminal sequence of the DC1 heavy chain. The heavy chain of the DC1 alloantigen is composed of 232 amino acids and can be divided into two external domains, alpha 1 (amino acids 1-87) and alpha 2 (amino acids 88-181), a connecting peptide (amino acids 182-194), a hydrophobic transmembrane region (amino acids 195-217), and an intracytoplasmic region (amino acids 218-232). Comparison with the HLA-DR heavy chain reveals strong sequence homology in the second external Ig-like domain (alpha 2) and the transmembrane region. In contrast, the first external domain, the connecting peptide, and the intracytoplasmic region are less conserved.