A haplotype of the human CXCR1 gene protective against rapid disease progression in HIV-1+ patients.

Chemokines and their receptors are key factors in the onset and progression of AIDS. Among them, accumulating evidence strongly indicates the involvement of IL-8 and its receptors, CXCR1 and CXCR2, in AIDS-related conditions. Through extensive investigation of genetic variations of the human CXCR1-CXCR2 locus, we identified a haplotype of the CXCR1 gene (CXCR1-Ha) carrying two nonsynonymous single nucleotide polymorphisms, CXCR1_300 (Met to Arg) in the N terminus extracellular domain and CXCR1_142 (Arg to Cys) in the C terminus intracellular domain. Transfection experiments with CXCR1 cDNAs corresponding to the CXCR1-Ha and the alternative CXCR1-HA haplotype showed reduced expression of CD4 and CXCR4 in CXCR1-Ha cells in human osteosarcoma cells as well as in Jurkat and CEM human T lymphocytes. Furthermore, the efficiency of X4-tropic HIV-1(NL4-3) infection was significantly lower in CXCR1-Ha cells than in CXCR1-HA cells. The results were further confirmed by a series of experiments using six HIV-1 clinical isolates from AIDS patients. A genetic association study was performed by using an HIV-1(+) patient cohort consisting of two subpopulations of AIDS with extreme phenotypes of rapid and slow progression of the disease. The frequency of the CXCR1-Ha allele is markedly less frequent in patients with rapid disease onset than those with slow progression (P = 0.0003). These results provide strong evidence of a protective role of the CXCR1-Ha allele on disease progression in AIDS, probably acting through modulation of CD4 and CXCR4 expression.

Exhaustive genotyping of the interferon alpha receptor 1 (IFNAR1) gene and association of an IFNAR1 protein variant with AIDS progression or susceptibility to HIV-1 infection in a French AIDS cohort.

We have undertaken a systematic genomic approach in order to explore the role of the interferon alpha (IFN-alpha) pathway in AIDS disease development. As it is very difficult to genotype the IFN-alpha gene itself since it has many pseudo-genes, we have focused our interest on the genetic polymorphisms of the IFN-alpha receptor 1 (IFNAR1). We genotyped the Genetics of Resistance to Immunodeficiency Virus (GRIV) cohort composed of patients with extreme profiles of progression to AIDS, slow progressors (SP) and rapid progressors (RP), as well as seronegative controls (CTR). We identified 19 single nucleotide polymorphisms (SNPs) with a minor allele frequency (MAF) greater than 1% among which two were newly characterized by our study. We found putative associations with AIDS disease development for four SNP alleles and for three haplotypes. The most interesting signals were found for two SNPs in linkage disequilibrium, the SNP IFNAR1_18339 corresponding to a Val168Leu mutation in the extracellular domain of the protein and the intronic SNP, IFNAR1_30127. The intronic SNP IFNAR1_30127 yielded a strong signal both when comparing SP with CTR (P=0.002) and RP with CTR (P=0.005) while IFNAR1_18339 yielded a smaller signal because less patients were analyzed; these SNPs could thus be involved in AIDS progression or in susceptibility to human immunodeficiency virus 1 (HIV-1) infection. Interestingly, two independent studies have previously pointed out the SNP IFNAR1_18339 in susceptibility to multiple sclerosis and to malaria. This is the first work investigating the polymorphisms of the IFNAR1 gene in AIDS. Our results which point out a possible role for the IFN-alpha pathway in susceptibility to HIV-1 infection or progression to AIDS need a necessary confirmation by genomic studies in other AIDS cohorts.

Active immunization against murine TNFalpha peptides in mice: generation of endogenous antibodies cross-reacting with the native cytokine and in vivo protection.

New lines of treatment targeting cytokines have been successfully developed recently and are now widely used in therapy. They are based on passive administration of cytokine inhibitors either soluble receptors or mAbs and the major example is TNFalpha in rheumatoid arthritis (RA). Since a few years, our group has developed a novel alternative approach targeting cytokines by using active immunization against biologically inactive but immunogenic cytokine derivatives. In the present work, we present a new aspect of this research, based on immunization against specific cytokine peptides chosen by molecular modelling. We could elicit a significant humoral response against four TNFalpha peptides by active immunization, and show that the Abs generated cross-reacted with the native cytokine with good titers as determined by ELISA. Interestingly, during coimmunization experiments with couples of peptides, one showed a clear immunodominant effect over the other. Overall, we could not show the neutralization of TNFalpha biological activity in vitro by the immunized sera, but it seems that it is not a prerequisite to observe clinical efficacy. Indeed, using the LPS/galactosamine-induced shock, we could demonstrate that one of the four peptides tested conferred a clinical protection. These results validate the feasibility and efficacy of active immunization against cytokine peptides, and confirm that active immunization against cytokines could represent in the future an alternative to passive immunization in many diseases.

Genomic analysis of Fas and FasL genes and absence of correlation with disease progression in AIDS.

Apoptosis has been suggested as a major mechanism for the CD4(+) T-lymphocyte depletion observed in patients infected with human immunodeficiency virus 1 (HIV-1). To evaluate the impact of genetic variations to apoptosis during progression of acquired immunodeficiency syndrome (AIDS), we have performed an extensive genetic analysis of Fas and Fas ligand ( FasL) genes. The coding regions and promoters of these genes were resequenced in a cohort of 212 HIV-1-seropositive patients presenting extreme disease phenotypes and 155 healthy controls of Caucasian origin. Overall, 33 single nucleotide polymorphisms (SNPs) with an allele frequency >1% were identified and evaluated for their association with disease progression. Among them, 14 polymorphisms were newly characterized. We did not find any statistically significant association of Fas and FasL polymorphisms and haplotypes with AIDS progression.

Apoptosis inhibition mediated by medroxyprogesterone acetate treatment of breast cancer cell lines.

Several reports suggested that steroidogenic hormones could be directly involved in the regulation of apoptosis in vitro, but whether this is due to blocking or promoting mechanism of these hormones remains controversial. However, it was shown that progesterone exhibited a protective effect against the apoptotic process during mouse mammary gland involution in vivo. In this study, we analyzed the effect of medroxyprogesterone acetate (MPA) treatment, an agonist of progesterone, on serum starvation induced apoptosis on breast cancer cell lines. Positive and negative progesterone receptor (PgR+ and PgR-) breast cancer cell lines were treated with MPA (10 nM), either in standard culture conditions or in serum-free medium to induce apoptosis. Cell survival, proliferation and apoptosis were simultaneously analyzed with the expression of apoptosis-related genes measured by a real time quantitative RT-PCR. At non cytotoxic doses, MPA protected PgR+ T47-D, MCF-7 and H466-B cell lines against serum depletion-induced apoptosis, while MPA did not protect PgR-MDA-MB-231 cells against serum depletion induced apoptosis. In PgR+ cell lines and in concordance with the protective effect, the pro-apoptotic HRK and BAK1 mRNAs were up-regulated after apoptosis induction, while they were no more induced in condition of protection against apoptosis after MPA treatment. We also observed, specifically in PgR+ cells, an up-regulation of BCLX-L and BCLX-S and a down-regulation of BCL2 mRNAs, which are specific to the MPA response and unrelated to apoptotic process. Involvement of these genes with regard to the MPA-mediated protection against apoptosis is discussed.

Proapoptotic effects of antiestrogens, progestins and androgen in breast cancer cells.

The promoting action of E2 in breast cancer cells has been, until now, mainly linked to its action on prolifieration. Because of the importance of an increase in apoptosis in breast cancer prevention, we have studied the possible effects of various antiestrogens, progestins and an androgen on its occurrence in three hormone-dependent breast cancer cell lines. The antiestrogens were, a triphenylethylene derivative, 4 hydroxytamoxifen (4OHTAM) and two steroidal antiestrogens, IC1182780 and RU58668. The progestins were Org2058, a pregnane derivative, tibolone (OrgOD14), a normethyltestosterone derivative and OrgOM38 (the delta4 isomer of OrgOD14) and the androgen dihydrotestosterone (DHT). Apoptosis was studied in MCF-7, ZR75-1 and T47-D cells using morphological approaches and flow cytometry. The antiestrogens, the progestins and DHT were proapoptotic but to different potencies according to the cell line studied. Indeed, the 'pure' steroidal antiestrogens were more efficient than 4OHTam in increasing apoptosis. We have also studied the level of expression of some of the proteins involved in the regulation of apoptosis. Bcl-2 and bcxL, two antiapoptotic members of the bcl-2 family proteins, were inhibited by the progestins and the antiestrogens. In contrast, the proapoptotic proteins, bax and bak seemed to be constitutively expressed. Thus, since the ratio of proapoptotic and antiapoptotic proteins determines apoptosis or cell survival, the hormone effects are operating by modulating the antiapoptotic regulators of the balance. These data demonstrate that antiestrogens, progestins, and androgens can promote apoptosis in breast cancer cells, an effect which could be of importance in the therapeutic prevention of breast cancer.

Increased prosomal proteins in breast cancer cells and in neighboring normal cells in Parsi and non-Parsi populations.

Monoclonal antibodies were raised against the prosomal proteins p27K, p29K and the prosome-like protein p21K (PLP) from normal breast glandular cells and from benign and malignant tumors. They were used to clarify the involvement of prosomes in tumorigenesis of human breast cells. Immunostaining showed the distribution of prosomes in the cytoplasm and nuclei of cells from European normal women (EN) and Parsi (P) and non-Parsi (NP) benign (B) and malignant (M) tissues. The flow-cytometry studies showed an increased mean percentage of labeled cells, particularly with anti-p27K prosomal protein mAb, in malignant tissue from NP compared to EN. The p21K data indicated an increase in the number of cells labeled by flow-cytometry studies in all groups compared to EN, while p29K-expressing cells were more abundant in NPN, PB, PM and NPM. Intergroup comparison showed that the mean percentage of cells labeled with anti-p27K and anti-p29K was significantly higher in PB than in NPB, as seen by flow cytometry, whereas there was a higher production or accumulation of the p21K (PLP) prosomal protein in NPM than in PM, as seen by immunostaining. By comparison with EN, there were also significantly more normal cells containing the three antigens in the apparently normal tissue in the neighborhood of the tumor in NPM, and more cells containing p21K in PM patients than in EN. As prosomes are involved in the cell differentiation and in the cell cycle control, the changes observed in breast tissues may be related to oncogenic processes. Furthermore, the modified subunit pattern of prosomes in cancer and, possibly, pre-cancer tissue may be of interest for diagnosis purposes.

Proliferation markers and cell cycle associated expression of prosomes in breast cancers of Parsis and non-Parsis.

The present study has focused attention essentially on the Parsis, an ethnic group with high breast cancer incidence. We have investigated the potential use of prosomes, compared to Ki-67 and PCNA, as an additional cell proliferation marker. We also addressed the question whether or not breast tumors of Parsis differed in their DNA index and in the proportion of the S-phase fraction, compared to that of non-Parsi and European patients. We observed that the benign tumors of Parsis and non-Parsis were hyperdiploids, whereas in case of malignant tumors the Parsis showed essentially diploid characteristics while hyperdiploidy prevailed in the non-Parsis. Tetraploidy was seen as a common feature in the non-Parsis, whereas aneuploidy seemed to be the more common type in the Parsis. The cell cycle analysis also revealed some interesting differences between the cell proliferation compartments of these two populations. A high number of cells in G2+M and S-phases was seen for non-Parsi malignant tumors while only the S-phase had a large cell count in the Parsis malignant tumors. The malignant tumors of Parsis and non-Parsis showed, as would be expected, a high expression of Ki-67 in the proliferation compartment. Surprisingly high Ki-67 expression was also a feature seen in the benign tumors of Parsis only and not any other group. We observed that expression of Ki-67, a proliferation marker directly related to the degree of malignancy, paralleled that of prosomal protein expression. In addition the prosomal monoclonal antibodies appeared to be more sensitive than Ki-67 in detecting a larger quantum of cells in the proliferation compartment.

p53 alterations in breast cancer of the Parsi ethnic group.

Mutations in p53 are the most common genetic abnormality yet found in human cancers. p53 mutations vary among tumor types, ranging from 0-60% in major cancers. The frequency of p53 mutations in breast cancer averages around 25%. The incidence rate and the type of mutations at specific codons seem to be influenced by geographical location as well as racial and ethnic specificities. Women of the Parsi ethnic group living mostly in defined geographical areas around metropolitan Bombay are reported to have a markedly high incidence of breast cancer. In the present work, the p53 gene alterations in the Parsis were investigated using SSCP analysis. The results confirm an earlier observation that over 60% of the Parsi breast tumors harbour p53 alterations. This figure is higher than that observed in other communities. The relevance of the finding in the light of the high breast cancer incidence in the Parsis is discussed.

Antagonism between estradiol and progestin on Bcl-2 expression in breast-cancer cells.

Bcl-2 is a key protein involved in the control of apoptosis. Our previous studies on breast and endometrium indicated hormonal regulation of bcl-2 in these tissues. In the present work we have analyzed Bcl-2 and Bax protein expressions in MCF-7 and T47-D, 2 hormone-dependent breast-cancer cell lines, by immunoblots. Estradiol markedly increased Bcl-2 protein content, both in short- and in long-term treatments of MCF-7 cells. Two types of anti-estrogens (4-hydroxytamoxifen and RU 58668) were able to reverse this effect. Also, a synthetic progestin (ORG 2058) was able to decrease the Bcl-2 level in T47-D cells. The level of Bax protein, however, was not affected in the same conditions of hormonal treatments. The level of Bcl-2 expression was 4.5-fold higher in MCF-7 than in MDA-MB 231 (an estradiol-independent cell line). From these results, we infer the existence of hormonal regulation of Bcl-2 expression and evoke a novel role for estradiol and progestin in the genesis of breast cancer.

bcl-2 proto-oncogene, apoptosis and oncogenesis.

Cell death has gained considerable interest during the last few years since, along with mitosis, it contributes to the determination of normal or neoplastic state of tissues. Cell death can occur in two ways: necrosis Or 'accidental' death and apoptosis or 'programmed' death. The focus on apoptosis is very recent. Like cell division it involves an interesting set of genetic regulations. The apoptosis-preventing bcl-2 gene, therefore emerges as a key-gene at the crossroad of cell death and cell proliferation: the two facets of the oncogenesis-governing equation.

Novel cellular markers in breast cancer.

Epidemiological studies have revealed that Parsi women have a higher incidence of breast cancer than non-Parsis and that they are more susceptible to breast cancer. We have studied the cellular distribution of two prosomal proteins p23K in parallel to the p30.33K and proliferation marker Ki-67 as potential markers to identify high risk population for breast cancers. Flow cytometry data demonstrated that the Parsi benign and non-Parsi malignants have a higher number of cells labelled with these two prosomal protein antibodies than the non-Parsi benign and European 'normals'. Using immunohistochemical methods, p23 K was found to be significantly higher in Parsi and non-Parsi malignants as well as in non-Parsi benigns. In our FCM analysis, intergroup comparison showed, interestingly, a significantly higher expression of both p23K and p30.33K in Parsi benigns as compared to non-Parsis, raising the possibility that benign tumors of Parsis represent already premalignant lesions. The present study, in addition, proposes the prosomal antigens as likely cell proliferation markers comparable to Ki-67.

Structural status of the int loci in women showing high incidence of breast cancer.

Mammary carcinomas in certain mice strains are induced following infection by the MMTV. Insertion of MMTV provirus into the int-1 and int-2 loci results in transcriptional activation of these two proto-oncogenes and is thought to be a key step in breast tumorigenesis in mice. A viral etiology for human breast cancers, though proposed several years ago, is far from proven. However, morphological structures resembling Bittner's particles have been observed earlier in about 39% samples of breast tumor and milk sediments of Parsi women. We therefore investigated in the Parsis, the structural integrity of two potential sites of integration (int-1 and int-2) of the hypothetical human mammary tumor virus and have used for this purpose a large number of patient DNA. The results obtained however failed to distinguish any major structural change existing at the int-1 or the int-2 sites that may point to a proviral integration event having occured in human breast cancers. We have, however, observed differences in the physical structure of the int-1 map, compared to the one reported and sequenced, and have therefore felt it necessary to present a new one indicating our findings, notably, the polymorphic PvuII sites. In addition, we report a single case of Parsi woman, with infiltrating grade 3 ductal carcinoma, whose DNA contains an alteration in the int-2 structure.

Bacteriophage lambda as a cloning vector.

Extensive research has been directed toward the development of multipurpose lambda vectors for cloning ever since the potential of using coliphage lambda as a cloning vector was recognized in the late 1970s. An understanding of the intrinsic molecular organization and of the genetic events which determine lysis or lysogeny in lambda has allowed investigators to modify it to suit the specific requirements of gene manipulations. Unwanted restriction sites have been altered and arranged together into suitable polylinkers. The development of a highly efficient in vitro packaging system has permitted the introduction of chimeric molecules into hosts. Biological containment of recombinants has been achieved by introducing amber mutations into the lambda genome and by using specific amber suppressor hosts. Taking advantage of the limited range of genome size (78 to 105% of the wild-type size) for its efficient packaging, an array of vectors has been devised to accommodate inserts of a wide size range, the limit being 24 kbp in Charon 40. The central dispensable fragment of the lambda genome can be replaced by a fragment of heterologous DNA, leading to the construction of replacement vectors such as Charon and EMBL. Alternatively, small DNA fragments can be inserted without removing the dispensable region of the lambda genome, as in lambda gt10 and lambda gt11 vectors. In addition, the introduction of many other desirable properties, such as NotI and SfiI sites in polylinkers (e.g., lambda gt22), T7 and T3 promoters for the in vitro transcription (e.g., lambda DASH), and the mechanism for in vivo excision of the intact insert (e.g., lambda ZAP), has facilitated both cloning and subsequent analysis. In most cases, the recombinants can be differentiated from the parental phages by their altered phenotype. Libraries constructed in lambda vectors are screened easily with antibody or nucleic acid probes since several thousand clones can be plated on a single petri dish. Besides the availability of a wide range of lambda vectors, many related techniques such as rapid isolation of lambda DNA, a high efficiency of commercially available in vitro packaging extracts, and in vitro amplification of DNA via the polymerase chain reaction have collectively contributed to lambda's becoming one of the most powerful and popular tools for molecular cloning.

Structure and expression of the chicken epidermal growth factor receptor gene locus.

Similarity between the carboxyl-terminal portion of the human epidermal growth factor (EGF) receptor and the deduced protein sequence of the chicken-derived oncogene v-erbB, of avian erythroblastosis virus strain H, has suggested that the chicken cellular erbB locus, c-erbB, might be part of a longer EGF-receptor gene in the chicken, whose entire coding capacity remained to be defined. The c-erbB locus spans more than 20 X 10(3) base pairs (20 kbp) of DNA and contains at least 1.8 kbp homologous to the v-erbB oncogene. We show here that human EGF receptor cDNA and chicken genomic DNA share homology not only within the c-erbB locus but also within a 25.1-kbp DNA region situated 5' to this locus. The 3' region of the EGF receptor overlaps, in sequence homology, the c-erbB locus. The EGF receptor/c-erbB locus in chicken generates six related but distinctly different mRNAs of sizes 12, 9, 5, 3.6, 3.2 and 2.6 kb. The transcripts of 12, 9, and 3.6 kb contain sequences coding for both the extracellular EGF-binding domain of the receptor and the intracellular tyrosine kinase domain. The 12-kb and 9-kb transcripts, which have already been shown to contain the sequences coding for the v-erbB, were found to possess, in addition, sequences that encode the entire chicken EGF receptor. The 3.2-kb and 2.6-kb mRNAs are homologous only to the 5' portion of the EGF receptor gene. These results therefore indicate that the c-erbB locus, initially defined by homology to the viral transforming gene, corresponds to the 3' region of the EGF receptor gene in the chicken genome. The multiple, related, chicken EGF receptor RNA transcripts reported here are reminiscent of the various human EGF receptor RNA transcripts observed in normal and transformed cells.

Altered globin gene transcription pattern and the presence of a 7-8 kb alpha A globin gene transcript in avian erythroblastosis virus-transformed cells.

Immature chick erythroid cells transformed by avian erythroblastosis virus (AEV) display an altered pattern of globin gene transcription leading to the abortive phenotypic expression of such transcripts. Detectable adult globin gene-specific RNA sequences, confined exclusively to the nucleus, are uniquely of the alpha A type. The alpha A globin-specific sequences occur in transcripts 7-8 kb long from which the 5' contiguous alpha D gene product is absent, and also in fragments smaller than 9S globin mRNA. The implication of this observation for schemes of post-transcriptional regulation of gene expression and viral transformation are discussed.