Nuclear localization and increased levels of transcription factor YB-1 in primary human breast cancers are associated with intrinsic MDR1 gene expression.

Breast cancers are either primarily resistant to chemotherapy (intrinsic resistance), or respond to chemotherapy but later recur with a multidrug-resistant phenotype because of overexpression of the multidrug transporter P-glycoprotein. The MDR1 gene encoding P-glycoprotein may be transcriptionally regulated by a Y-box transcription factor. We now report that, in multidrug-resistant MCF-7 breast cancer cells, nuclear localization of YB-1 is associated with MDR-1 gene expression. In drug-sensitive MCF-7 cells, however, YB-1 was localized to the cytoplasm. Regulated overexpression of YB-1 in drug-sensitive diploid breast epithelial cells induced MDR-1 gene expression and multidrug resistance. In 27 out of 27 untreated primary breast cancers, YB-1 protein was expressed in the cytoplasm although it was undetectable in normal breast tissue of these patients. In a subgroup of tumors (9/27), however, YB-1 was also localized to the nucleus and, in these cases, high levels of P-glycoprotein were present. These results show that in a subset of untreated primary breast cancers, nuclear localization of YB-1 protein is associated with intrinsic multidrug resistance. Our data show that YB-1 has an important role in controlling MDR1 gene transcription and this finding provides a basis for the analysis of molecular mechanisms responsible for intrinsic multidrug resistance in human breast cancer.

Functional isotypes are not encoded by the constant region genes of the beta subunit of the T cell receptor for antigen/major histocompatibility complex.

Human T cell clones and a cDNA probe specific for constant regions of the beta subunit of the antigen/major histocompatibility complex (MHC) receptor, TiC beta 1 and TiC beta 2, were employed to determine whether these genes were differentially used by functional classes of T lymphocytes. DNA from 10 interleukin-2-dependent T cell clones including class I and class II MHC-specific cytotoxic T lymphocytes (n = 6), T4+ inducer T lymphocytes (n = 2), and T8+ suppressor T lymphocytes (n = 2) showed rearrangement of the TiC beta 1 gene on Southern blot analysis with or without deletion of the other TiC beta 1 allele. In contrast, TiC beta 2 always remained in germline configuration. Moreover, the finding that one additional suppressor clone deleted both TiC beta 1 alleles, maintained a germline TiC beta 2 configuration, and yet actively transcribed TiC beta 2 message suggested that TiC beta 2 is not a pseudogene. Rather, it appeared to be used less frequently than the TiC beta 1 gene and in the absence of detectable DNA rearrangements. Together, these results demonstrate that the functional repertoire (or isotype) of a given subclass of T cells is not encoded within the Ti beta genes.

Regional location of T cell receptor gene Ti alpha on human chromosome 14.

The chromosomal location of Ti alpha was determined by hybridization of a radiolabeled cDNA for the alpha chain of human T cell receptor with 12 human X mouse cell hybrid DNAs cleaved with BamHI. Seven hybrids contained human Ti alpha, while the remaining five lacked it. Only human chromosome 14 matched the distribution of human Ti alpha signal across the mapping panel. Hybrids segregating a chromosome 14 translocation were used to demonstrate that Ti alpha is in the region 14pter greater than 14q21. Thus, the alpha and beta chain genes that contribute structural components to the Ti moiety of the human T cell receptor lie on different chromosomes. In humans, the immunoglobulin heavy chain locus and Ti alpha are in different regions of chromosome 14, with Ti alpha more proximal and the immunoglobulin heavy chain locus more distal.

Molecular analysis of T cell receptor (Ti) variable region (V) gene expression. Evidence that a single Ti beta V gene family can be used in formation of V domains on phenotypically and functionally diverse T cell populations.

We examine the rules governing Ti beta variable (V) gene segment usage in the formation of T cell antigen-MHC receptors in diverse regulatory and effector T lymphoid subpopulations. To this end, a single Ti beta V gene family and its products were analyzed. A monoclonal antibody, termed anti-Ti3A, which was shown to be reactive with an epitope encoded by members of the REX cell line Ti beta V gene family, and which was expressed on 2% of human T lymphocytes was used in selection of clones from unprimed peripheral T lymphocytes. Both T4+, as well as T8+ T cell clones with inducer, suppressor, and/or cytotoxic function were defined. Southern analysis, isoelectric focusing and two-dimensional peptide mapping indicated that individual members of the REX V gene family were linked to different Ti beta diversity and/or joining and constant region segments. Moreover, the Ti alpha chains of such clones were distinct. These results imply that Ti beta V gene usage is not restricted to any functionally or phenotypically defined T cell subsets, and there is presumably little, if any, restriction on the mechanisms that generate combinational, junctional or chain association-mediated diversity.

Expression of the NKTa clonotype in a series of human natural killer clones with identical cytotoxic specificity.

Over a period of 3 yr, a series of ten NK clones that express a unique clonotypic T cell receptor-like structure, termed NKTa, has been generated from a single individual. These clones were derived from either peripheral blood nonadherent cell fractions (JT9, JT10, JT11), NKH2-purified cells (CNK8, CNK9), or NKTa-purified cells (CNK11, CNK12, CNK13, CNK14, CNK15). Flow cytometric analysis of peripheral blood mononuclear cells from this individual showed that NKTa+ cells occur with a frequency of approximately 0.15%. The existence of NKTa+ cells in peripheral blood was confirmed by use of immunorosette enrichment techniques, flow cytometric purification, and subsequent clonal expansion of NKTa+ cells. Phenotypic analysis of NKTa+ clones showed that all expressed NKH1 as well as T3, T8, T11, T12, and Mo1 antigens. Only five of ten clones expressed NKH2 antigen. All NKTa+ clones had broad cytolytic activity against a series of seven different target cells that was similar to that of other NK clones. In addition, cytotoxicity of each clone could be inhibited by preincubation of effector cells with monoclonal anti-NKTa or by preincubation of target cells with monoclonal anti-TNKTAR. Although half of the NKTa+ clones appeared phenotypically different from the other half with regard to the expression of NKH2 antigen, analysis of T cell receptor gene rearrangements indicated that all NKTa+ clones contained identical gene rearrangements of C beta 2.

Blocking the transcription factor E2F/DP by dominant-negative mutants in a normal breast epithelial cell line efficiently inhibits apoptosis and induces tumor growth in SCID mice.

The transcription factor E2F is regulated during the cell cycle through interactions with the product of the retinoblastoma susceptibility gene and related proteins. It is thought that E2F-mediated gene regulation at the G1/S boundary and during S phase may be one of the rate-limiting steps in cell proliferation. It was reported that in vivo overexpression of E2F-1 in fibroblasts induces S phase entry and leads to apoptosis. This observation suggests that E2F plays a role in both cell cycle regulation and apoptosis. To further understand the role of E2F in cell cycle progression, cell death, and tumor development, we have blocked endogenous E2F activity in HBL-100 cells, derived from nonmalignant human breast epithelium, using dominant-negative mutants under the control of a tetracycline-dependent expression system. We have shown here that induction of dominant-negative mutants led to strong downregulation of transiently transfected E2F-dependent chloramphenicol acetyl transferase reporter constructs and of endogenous c-myc, which has been described as a target gene of the transcription factor E2F/DP. In addition, we have shown that blocking of E2F could efficiently protect from apoptosis induced by serum starvation within a period of 10 d, whereas control cells started to die after 24 h. Surprisingly, blocking of E2F did not alter the rate of proliferation or of DNA synthesis of these cells; this finding indicates that cell-cycle progression could be driven in an E2F-independent manner. In addition, we have been able to show that blocking of endogenous E2F in HBL-100 cells led to rapid induction of tumor growth in severe combined immunodeficiency mice. No tumor growth could be observed in mice that received mock-transfected clones or tetracycline to block expression of the E2F mutant constructs in vivo. Thus, it appears that E2F has a potential tumor-suppressive function under certain circumstances. Furthermore, we provide evidence that dysregulation of apoptosis may be an important step in tumorigenesis.

Chromosomal location of human T-cell receptor gene Ti beta.

A complementary DNA probe corresponding to the beta-chain gene of Ti, the human T lymphocyte receptor, has been molecularly cloned. The chromosomal origin of the Ti beta gene was determined with the complementary DNA by screening a series of 12 cell hybrid (mouse X human) DNA's containing overlapping subsets of human chromosomes. DNA hybridization (Southern) experiments showed that the human Ti beta gene resides on chromosome 7 and is thus not linked to the immunoglobulin loci or to the major histocompatibility locus in humans.

Analysis of T-cell receptor gene rearrangement and expression in human natural killer clones.

A series of clones of human natural killer (NK) cells was characterized with respect to expression of the Ti alpha and Ti beta genes of the T-cell receptor. T11+T3+ NK clones contained Ti alpha and Ti beta RNA transcripts and expressed disulfide-linked heterodimers, demonstrating the presence of a functional T-cell receptor. In contrast, T11+T3- NK clones expressed only 1.0-kilobase truncated Ti beta transcripts, without a Ti alpha transcript and no detectable surface Ti protein. Since previous studies demonstrated that Ti beta gene activation precedes Ti alpha gene activation in thymic ontogeny, the T11+T3- NK cells appear to be derived from T-lineage precursors.

Constitutive nuclear factor-kappaB-RelA activation is required for proliferation and survival of Hodgkin's disease tumor cells.

The pathogenesis and etiology of Hodgkin's disease, a common human malignant lymphoma, is still unresolved. As a unique characteristic, we have identified constitutive activation of the transcription factor nuclear factor (NF)-kappaB p50-RelA in Hodgkin/Reed-Sternberg (H/RS) cells, which discriminates these neoplastic cells from most cell types studied to date. In contrast to other lymphoid and nonlymphoid cell lines tested, proliferation of H/RS cells depended on activated NF-kappaB. Furthermore, constitutive NF-kappaB p50-RelA prevented Hodgkin's lymphoma cells from undergoing apoptosis under stress conditions. Consistent with this dual function, Hodgkin's lymphoma cells depleted of constitutive nuclear NF-kappaB revealed strongly impaired tumor growth in severe combined immunodeficient mice. Our findings identify NF-kappaB as an important component for understanding the pathogenesis of Hodgkin's disease and for developing new therapeutic strategies against it.

Overexpression of the death-promoting gene bax-alpha which is downregulated in breast cancer restores sensitivity to different apoptotic stimuli and reduces tumor growth in SCID mice.

We have studied the expression of members of the bcl-2 family in human breast cancer. The expression pattern of these genes in breast cancer tissue samples was compared with the expression pattern in normal breast epithelium. No marked difference with regard to bcl-2 and bcl-xL expression was observed between normal breast epithelium and cancer tissue. In contrast, bax-alpha, a splice variant of bax, which promotes apoptosis, is expressed in high amounts in normal breast epithelium, whereas only weak or no expression could be detected in 39 out of 40 cancer tissue samples examined so far. Of interest, downregulation of bax-alpha was found in different histological subtypes. Furthermore, we transfected bax-alpha into breast cancer cell lines under the control of a tetracycline-dependent expression system. We were able to demonstrate for the first time that induction of bax expression in breast cancer cell lines restores sensitivity towards both serum starvation and APO-I/Fas-triggered apoptosis and significantly reduces tumor growth in SCID mice. Therefore, we propose that dysregulation of apoptosis might contribute to the pathogenesis of breast cancer at least in part due to an imbalance between members of the bcl-2 gene family.

Uncoupling of EGFR-RAS signaling and nuclear localization of YBX1 in colorectal cancer.

The transcription factor YBX1 can act as a mediator of signals transmitted via the EGFR-RAS-MAPK axis. YBX1 expression has been associated with tumor progression and prognosis in multiple types of cancer. Immunohistochemical studies have revealed dependency between YBX1 expression and individual EGFR family members. We analyzed YBX1 and EGFR family proteins in a colorectal cancer (CRC) cohort and provide functional analyses of YBX1 in the context of EGFR-RAS-MAPK signaling. Immunohistochemistry for YBX1 and EGFR family receptors with two antibodies for YBX1 and EGFR were performed and related to clinicopathological data. We employed Caco2 cells expressing an inducible KRASV12 gene to determine effects on localization and levels of YBX1. Mouse xenografts of Caco2-KRASV12 cells were used to determine YBX1 dynamics in a tissue context. The two different antibodies against YBX1 showed discordant immunohistochemical stainings in cell culture and clinical specimens. Expression of YBX1 and EGFR family members were not correlated in CRC. Analysis of Caco2 xenografts displayed again heterogeneity of YBX1 staining with both antibodies. Our results suggest that YBX1 is controlled via complex regulatory mechanisms involving tumor stroma interaction and signal transduction processes. Our study highlights that YBX1 antibodies have different specificities, advocating their use in a combined manner.

Characterization of the transcriptional regulatory region of the human WT1 gene.

Specific control of the expression of the Wilms tumor gene WT1 is important for normal development of the kidney. In order to characterise the transcriptional control region of the WT1 gene we have isolated genomic clones spanning the upstream region, the first WT1 exon and the 5' end of the Wit1 gene. DNA sequencing revealed that the WT1 promoter lacks a TATA box or CCAAT motif and has a GC content of 71%. Four transcriptional start sites are clustered within a 32 bp region. GC-boxes are present at nucleotide positions -413, -160, +84 and +158. DNAase I protection assays with purified Sp1 protein revealed the existence of 11 different binding sites in the WT1 promoter. WT1 and Wit1 promoter activities were tested in COS-7 cells with luciferase reporter gene constructs either containing or lacking an SV40 enhancer. WT1 promoter activity was found in a fragment extending from 449 bp upstream to 201 bp downstream of the WT1 start site. It was 26 fold lower in the absence of the SV40 enhancer than in the presence. Cotransfection with a Sp1 expression vector stimulated both constructs 3-4 fold. Wit1 promoter activity was identified in a DNA fragment extending from 200 bp upstream of the putative Wit1 TATA box to 130 bp downstream. Several potential recognition sites for WT1/EGR, Pax-8, and GAGA-like transcription factors are present in the WT1 promoter.

Synthesis of high molecular weight polypeptides in Escherichia coli minicells directed by cloned Saccharomyces cerevisiae 2-micron DNA.

The minicell-producing Escherichia coli strain P 678-54 was transformed with a series of defined PTY chimeric plasmids consisting of yeast 2-micron DNA and E. coli plasmid pCR1. In minicells the integrated 2-micron DNA from yeast directed specifically the synthesis of six polypeptides with apparent molecular weights of 15,000, 17,500, 20,000, 22,000, 37,000, AND 48,000. The specificity of five other polypeptides, which cover a molecular weight range of 19,000 to 28,000, has not yet been established with certainty. Neither the orientation of the integrated DNA, nor the inversion which distinguishes the two structural forms of 2-micron DNA affected the polypeptides synthesized. However, integration at a given EcoRI site appeared to be correlated with the absence of one particular polypeptide band; this suggests that at least one of these sites is located in an expressed region of the DNA.

The ontogeny, structure and function of the human T lymphocyte receptor for antigen and major histocompatibility complex.

Recent studies using cloned antigen-specific T lymphocytes and monoclonal antibodies directed at their various surface glycoprotein components have led to identification of the human T cell antigen receptor as a surface complex comprised of a clonotypic 90 kDa Ti heterodimer and the invariant 20 and 25 kDa T3 molecules. Approximately 30,000-40,000 Ti and T3 molecules exist on the surface of human T lymphocytes. These glycoproteins are acquired and expressed during late thymic ontogeny, thus providing the structural basis for immunological competence. The alpha and beta subunits of Ti bear no precursor-product relationship to one another and are encoded by separate germline V, D, J and C segments which rearrange during intrathymic differentiation to form an active gene set. Triggering of the T3-Ti receptor complex induces a rapid increase in free cytoplasmic Ca2+ and gives rise to specific antigen-induced proliferation through an autocrine pathway involving endogenous interleukin-2 production, release and subsequent binding to interleukin-2 receptors. The implications of these findings for understanding of human T cell growth and its regulation in disease states are discussed.

Multiple octamer-binding proteins are targets for the cell cycle-regulated nuclear inhibitor I-92.

p92 is a novel sequence-specific octamer-binding factor interacting with the enhancer of human papillomavirus type 18. The nuclear inhibitor I-92 regulates the DNA binding activity of p92 during the cell cycle such that p92 DNA binding is restricted to S-phase. The sequence motif ++ 5'-AATTGCTTGCATAA, consisting of two partially overlapping octamer-related sequences, represents a recognition site for p92. It was the aim of this study to characterize the complexity of proteins interacting with the 5'-AATTGCTTGCATAA motif and to determine their regulation by I-92. UV cross-linking experiments showed that, besides p92, multiple novel proteins interact with the 5'-AATTGCTGCATAA motif. These novel proteins p84, p75, p73, p69, p61, p57, p49, and p46 specifically bind to this motif, although with different affinities. The inhibitor I-92 regulates, besides p92, the DNA-binding activities of p84, p75, p73, p69, and p57 but not of p61, p49, and p46. The association of I-92 with p92, p84, p75, p73, p69, and p57 was completely reversible after treatment with the detergent deoxycholate (DOC). Finally, we analyzed I-92 specificity and found that I-92 selectively inhibited DNA binding activities of partially purified octamer-binding proteins p84 and p92 whereas DNA binding of the POU factor Oct-1 was not regulated by I-92. Our results show that I-92 regulates multiple octamer-binding proteins and these findings provide an example how gene regulation could be linked to cell cycle regulation.

Multiple nuclear proteins bind upstream sequences in the promotor region of a T-cell receptor beta-chain variable-region gene: evidence for tissue specificity.

DNA-nuclear protein binding interactions were examined in the promoter region of a representative T-cell receptor Ti beta-chain variable-region gene by means of electrophoretic mobility-shift and DNase I-protection analysis. Within 175 bases upstream of the transcription initiation site, four protected regions ("footprints") were identified on the coding strand, at nucleotides -46 to -68 (I), -72 to -92 (II), -113 to -134 (III), and -136 to -175 (IV). Nuclear proteins (0.6 M NaCl fraction from a heparin-Sepharose column chromatography of nuclear extracts) of a variety of cell types produced footprints I, III, and IV and a fifth footprint (beyond nucleotide -200). In contrast, footprint II was produced only by T-cell extracts, although nuclear extracts of a transformed B-lymphoblastoid line produced a partial footprint in this region. Furthermore, footprint analysis of the noncoding strand showed that a continuous region of protection corresponding to the entire region of footprints I and II was generated, along with a DNase I-hypersensitive site, by nuclear proteins derived from T cells but not other cell types. Footprint I has the sequence structure of many well-defined protein-DNA binding sites. Nucleotide sequences in the region of footprint II bore no homology to known sequences, whereas those in the areas of footprints III and IV were similar to motifs within immunoglobulin and other enhancers. These findings may have implications for the tissue specificity of human Ti beta-chain gene transcription.

The human T cell receptor for antigen: structure, ontogeny and gene expression.

T cell receptor molecules are now well characterized as well as the genes encoding alpha and beta chains of this molecular complex. The genome organisation of alpha and beta chain genes is similar to the genomic organisation of immunoglobulin genes. In T cell differentiation immature precursor cells move into the human thymus, where they mature and subsequently are released into the periphery as immuno-competent T cells with a variety of different functions. The processes at work in thymic ontogeny are understood, only in part, and await further study. One aspect of T cell differentiation is the acquisition of immunocompetence by T cells in thymic ontogeny. This process is associated with T cell receptor gene rearrangements and T cell receptor gene expression. The mechanisms leading to gene expression have been studied in many systems and basic principles are now emerging. The enzyme RNA polymerase II, which synthesizes m-RNA requires additional factors for its activity, and these factors have, at least in part, been identified as proteins. Experiments aimed at identifying DNA-protein interactions at the V beta upstream regulatory region are discussed.

Methylation of chloroplast DNAs in the life cycle of Chlamydomonas.

Methylation patterns of Chlamydomonas chloroplast DNAs (chlDNAs) were examined in the vegetative, gametic, and zygotic stages of the life cycle. Restriction endo-nuclease fragment patterns produced by EcoRI, BamHI, Hpa II, and Msp I were compared; the last two cleave DNA at the sequence C-C-G-G, but Hpa II is blocked by prior methylation of the internal cytidine whereas Msp I is not. chlDNAs from vegetative cells of both mating types showed no evidence of methylation at C-C-G-G. Gametic mt+ chlDNA was heavily methylated at C-C-G-G, whereas the homologous chlDNA from mt- gametes showed very slight methylation at C-C-G-G. Methylation of additional sites in chlDNA from mt+ gametes but not from mt- gametes was shown by blockage of some EcoRI and BamHI sites that were cleaved in the chlDNA from vegetative cells. chlDNA from 6-hr zygotes was much more methylated than gametic mt+ DNA, as shown by its almost total resistance to cleavage by all four restriction enzymes. These findings support and extend previous evidence that chlDNA of mt+ cells is methylated during gametogenesis and that further methylation occurs after gametic fusion in the young zygotes.