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Biomedical subjects

G Blanck

Publications and source records attributed to G Blanck.

At least 19 recordsLinked to original sources
PubMed2001

Loss of MHC class II inducibility in hyperplastic tissue in Rb-defective mice.

Retinoblastoma gene (Rb) defects occur frequently in human tumors. Studies of Rb-defective human tumor cell lines and Rb-/- murine embryonic fibroblasts demonstrate that Rb is required for interferon-gamma (IFN-gamma) induced major histocompatibility complex (MHC) class II expression. MHC class II expressing tumors generate anti-tumor immune responses associated with tumor-specific infiltrating lymphocytes. The role of Rb in IFN-gamma induced MHC class II expression on an endogenous tumor was examined by immunohistochemical staining for IAbeta and Rb on tissues from Rb+/- mice. MHC class II IAbeta is not induced by IFN-gamma in Rb-deficient neoplastic cells, but remains inducible in related normal tissue.

Animals↗
PubMed2001

Impaired class II transactivator expression in mice lacking interferon regulatory factor-2.

Class II transactivator (CIITA) is required for both constitutive and inducible expression of MHC class II genes. IFN-gamma induced expression of CIITA in various cell types is directed by CIITA type IV promoter. The two transactivators, STAT1 and IRF-1, mediate the IFN-gamma activation of the type IV promoter by binding to the GAS and IRF-E of the promoter, respectively. In addition to IRF-1, IRF-2, another member of the IRF family, also activates the human CIITA type IV promoter, and IRF-2 cooperates with IRF-1 to activate the promoter in transient transfection assays. IRF-1 and IRF-2 can co-occupy the IRF-E of the human CIITA type IV promoter. To understand the effect of loss of IRF-2 on the endogenous CIITA expression, we assayed for CIITA expression in IRF-2 knock-out mice. Both basal and IFN-gamma induced CIITA expression were reduced in IRF-2 knock-out mice. At least half of the amount of inducible CIITA mRNA depends on IRF-2. The reduction of IFN-gamma induced CIITA mRNA in IRF-2 knock-out mice was due to the reduction of the type IV CIITA mRNA induction. The reduction of basal CIITA mRNA was apparently due to the reduction of CIITA mRNA originating from other promoters. These data indicate that IRF-2, like IRF-1, plays a critical role in the regulation of the endogenous CIITA gene. The implications in understanding the previously described phenotypes of IRF-2 defective mice are discussed.

Animals↗
PubMed2001

High level class II trans-activator induction does not occur with transient activation of the IFN-gamma signaling pathway.

Gene activation in early development is highly dependent on precise concentrations of trans-acting factors for the activation of different genes at differing points in the embryo. Thus, not only is the presence or absence of a particular trans-activator or repressor relevant in determining gene activation, but also the concentration of the regulatory protein must be above or below a certain threshold for proper gene regulation. Signaling pathways in somatic cells are thought to represent cascades of on/off switches, mediated most commonly by phosphorylation. Here we demonstrate a quantitative mechanism for regulating the level of a component of the IFN-gamma signaling pathway that in effect represents the differential sensitivities of STAT1, IFN-regulatory factor-1, and class II trans-activator (CIITA) to IFN-gamma. Unlike developmental gene regulation, in which specificity of gene activation is a function of regulatory protein concentrations, specificity of gene activation in the IFN-gamma signaling pathway is regulated by the duration of the activation of the primary IFN-gamma-regulatory protein, STAT1. This result most likely explains previously reported data indicating that a minimum amount of IFN-gamma is required for MHC class II gene activation despite the fact that the level of the IFN-gamma-inducible factor directly required for MHC class II induction, CIITA, directly correlates with the level of MHC class II expression. The induction of a high level of CIITA is dependent on sustained IFN-gamma signaling. The possible implications of this result for tumorigenesis are discussed.

Cell Line, Transformed↗
PubMed2001

Histone deacetylase activity represses gamma interferon-inducible HLA-DR gene expression following the establishment of a DNase I-hypersensitive chromatin conformation.

Expression of the retinoblastoma tumor suppressor protein (Rb) is required for gamma interferon (IFN-gamma)-inducible major histocompatibility complex class II gene expression and transcriptionally productive HLA-DRA promoter occupancy in several human tumor cell lines. Treatment of these Rb-defective tumor cell lines with histone deacetylase (HDAC) inhibitors rescued IFN-gamma-inducible HLA-DRA and -DRB mRNA and cell surface protein expression, demonstrating repression of these genes by endogenous cellular HDAC activity. Additionally, Rb-defective, transcriptionally incompetent tumor cells retained the HLA-DRA promoter DNase I-hypersensitive site. Thus, HDAC-mediated repression of the HLA-DRA promoter occurs following the establishment of an apparent nucleosome-free promoter region and before transcriptionally productive occupancy of the promoter by the required transactivators. Repression of HLA-DRA promoter activation by HDAC activity likely involves a YY1 binding element located in the first exon of the HLA-DRA gene. Chromatin immunoprecipitation experiments localized YY1 to the HLA-DRA gene in Rb-defective tumor cells. Additionally, mutation of the YY1 binding site prevented repression of the promoter by HDAC1 and partially prevented activation of the promoter by trichostatin A. Mutation of the octamer element also significantly reduced the ability of HDAC1 to confer repression of inducible HLA-DRA promoter activation. Treatment of Rb-defective tumor cells with HDAC inhibitors greatly reduced the DNA binding activity of Oct-1, a repressor of inducible HLA-DRA promoter activation. These findings represent the first evidence that HDAC activity can repress IFN-gamma-inducible HLA class II gene expression and also demonstrate that HDAC activity can contribute to promoter repression following the establishment of a DNase I-hypersensitive chromatin conformation.

Butyrates↗
PubMed2001

Varying functions of specific major histocompatibility class II transactivator promoter III and IV elements in melanoma cell lines.

Melanoma cells commonly express MHC class II molecules constitutively. This is a rare, or possibly unique, phenotype for a nonprofessional antigen-presenting cell, where MHC class II expression ordinarily occurs only after IFN-gamma treatment. Despite the fact that constitutive expression of MHC class II on melanoma cells has been observed for decades and that the regulation of the MHC class II genes is well understood for many different cell types, there is no data regarding the basis for constitutive MHC class II expression in melanoma cells. Here we report that MHC class II expression in melanoma cells can be traced to constitutive expression of the class II transactivator protein (CIITA), which mediates both IFN-gamma-inducible and -constitutive MHC class II expression in all other cell types. In addition, we determined that constitutive CIITA expression is the result of the activation of both the B cell-specific CIITA promoter III and the IFN-gamma-inducible CIITA promoter IV, the latter of which previously has never been known to function as a constitutive promoter in any cell type. The recently described B cell-related ARE-1 activity is important for promoter III activation in the melanoma cells. Constitutive promoter IV activation involves the IFN regulatory factor element (IRF-E), which binds members of the IRF family of proteins, although the major, IFN-gamma inducible member of this family, IRF-1, is not constitutively expressed in these cells. In cells with constitutively active promoter IV, the promoter IV IRF-E is most likely activated by IRF-2. The relevance of these results to the pathway of melanoma development is discussed.

DNA-Binding Proteins↗
PubMed2000

Interferon regulatory factor-2 point mutations in human pancreatic tumors.

Interferon regulatory factor (IRF)-2, a member of the IRF family, is a transcription factor involved in the regulation of various interferon and virus-stimulated genes and other genes. For example, IRF-2 is an activator of the interferon (IFN)-gamma-inducible MHC class II transactivator (CIITA) type IV promoter. It cooperates with IRF-1 in the activation of the CIITA type IV promoter and can co-occupy the IRF-E of the promoter with IRF-1. In a previous study, we identified an inactivating point mutation in the DNA binding domain of IRF-2 expressed in a human pancreatic tumor cell line that does not express CIITA or MHC class II in response to IFN-gamma. To further assess the potential impact of IRF-2 mutations in tumorigenesis, we screened fresh pancreatic tumor explants and identified 2 IRF-2 point mutations in the 2 alleles of IRF-2 from a single tumor specimen. Both mutations occurred in the DNA binding domain of IRF-2. DNA binding assays demonstrated that the IRF-2 point mutations impaired IRF-2 DNA binding. The transactivation function of the mutant IRF-2s was similarly impaired. This is the first report of IRF-2 mutations in human tumor explants.

Alleles↗
PubMed2000

Retinoblastoma protein activation of interleukin 8 expression inhibits tumor cell survival in nude mice.

Loss of retinoblastoma protein (Rb) has been implicated in the formation of a variety of human malignancies. Restoration of Rb expression in the cell lines representing these tumors eliminates or significantly reduces tumorigenicity in nude mice, but the mechanism for this Rb effect is unknown. Results from this study indicated that Rb expression reduced tumor cell survival in nude mice by dramatically enhancing interleukin 8 (IL-8) secretion. IL-8 secreted by the Rb-transformed cells attracted neutrophils in vitro and tumor-infiltrating neutrophils in vivo, which is consistent with the Rb-mediated tumor regression being dependent on IL-8. The apparent, contradictory roles of IL-8 as a protumorigenic and antitumorigenic cytokine are discussed.

Animals↗
PubMed1999

Co-occupancy of the interferon regulatory element of the class II transactivator (CIITA) type IV promoter by interferon regulatory factors 1 and 2.

Class II transactivator (CIITA) activates the expression of major histocompatibility class II genes, which encode antigen-presenting molecules recognized by the T-cell receptor of CD4+ T cells. IFN-gamma induced CIITA transcription in many cell types is directed by the CIITA Type IV promoter. Here we report that the human CIITA Type IV promoter IRF-E binds IRF-1 and can be activated by exogenous expression of IRF-1. Surprisingly, the CIITA Type IV promoter IRF-E is also activated by IRF-2, another member of the IRF family that generally acts as a transcriptional repressor. In addition, we found that IRF-1 and IRF-2 synergistically activate the CIITA Type IV promoter. Electrophoretic mobility shift assays revealed that IRF-1 and IRF-2 can simultaneously occupy the IRF-E of the CIITA Type IV promoter, suggesting a novel mechanism for the role of these two proteins in promoter activation. Our results also indicate that IRF-1 and IRF-2 can cooperatively activate and co-occupy the IRF-E of the guanylate binding protein (GBP) promoter. Finally, CIITA induction by IFN-gamma does not occur in a pancreatic tumor cell line that expresses a mutated IRF-2, representing the first IRF-2 mutation identified in a human tumor cell line.

DNA-Binding Proteins↗
PubMed1999

Interferon regulatory factor 1 tryptophan 11 to arginine point mutation abolishes DNA binding.

Interferon regulatory factor-1 (IRF-1) is a transcriptional activator of genes induced by a variety of cytokines and growth factors. Defects in IRF-1 occur frequently in human cancers and may contribute to tumorigenesis. The IRF family of transcription factors share invariant tryptophan residues that have been proposed to function by orienting the DNA contacting residues of IRF-1 with the DNA core sequence of the IRF element. Here we describe a point mutation in IRF-1 that converts the tryptophan at codon 11 to arginine (W11R). The IRF-1 (W11R) mutation abolishes IRF-1 DNA binding and transactivating activities demonstrating the critical role of this invariant tryptophan in IRF-1 function.

Arginine↗
PubMed1999

Retinoblastoma protein expression leads to reduced Oct-1 DNA binding activity and enhances interleukin-8 expression.

Tumor cell lines with a defective retinoblastoma gene are unable to transcribe the HLA class II genes in response to IFN-gamma treatment, and reconstitution of functional Rb rescues IFN-gamma-induced class II gene expression. However, the molecular mechanism of Rb rescue of the class II genes is unknown. We have examined the effect of Rb expression on the activation of the promoter for HLA-DRA, the prototype class II gene. Oct-1, a POU domain transcription factor, was identified as a repressor of HLA-DRA promoter activity in the Rb-defective cells. Rb expression led to phosphorylation of Oct-1, thus relieving its repressive effect. Oct-1 has also been shown to repress interleukin 8 promoter activity. Consistent with reduced levels of Oct-1 DNA binding activity in the Rb-transformed cell lines, interleukin 8 expression is higher in these cell lines.

Blotting, Western↗
PubMed1997

Retinoblastoma protein expression facilitates chromatin remodeling at the HLA-DRA promoter.

The major histocompatibility complex (MHC) class II genes encode a series of heterodimeric cell surface glycoproteins that bind peptide antigen. The MHC class II/peptide complex is bound by the T-cell receptor of CD4(+) T cells, thereby stimulating an immune response. The MHC class II genes are coordinately regulated by conserved promoter elements and are inducible by IFN-gamma. Furthermore, IFN-gamma induction of the MHC class II genes in solid human tumor lines requires retinoblastoma protein (Rb). In vivo footprinting analyses of the HLA-DRA gene, which encodes the heavy chain subunit of the human MHC class II molecule, HLA-DR, revealed that Rb facilitates occupancy of multiple HLA-DRA promoter elements. Detecting the effect of Rb on HLA-DRA promoter occupancy in vivo required IFN-gamma treatment. However, use of a variation on the in vivo footprinting technique, nuclei footprinting, which assays for promoter occupancy in isolated nuclei, revealed that expression of Rb facilitates promoter occupancy even in the absence of IFN-gamma. These results indicate that expression of Rb leads to modification of the chromatin environment of the HLA-DRA promoter independently of transcription.

Chromatin↗
PubMed1997

IFN-gamma inducibility of class II transactivator is specifically lacking in human tumour lines: relevance to retinoblastoma protein rescue of IFN-gamma inducibility of the HLA class II genes.

We have previously reported that HLA class II induction by IFN-gamma is rescuable by reconstitution of functional retinoblastoma protein (RB) in two RB-defective tumour lines: the breast carcinoma line, MDA-468-S4 (S4) and the non-small cell lung carcinoma line, H2009. To determine the range of tumours and tumour types in which RB rescues HLA class II inducibility, we examined another RB-defective tumour line, the retinoblastoma line, WERI-Rb1. As in the case of S4 and H2009, HLA-DRA and -DRB were non-inducible by IFN-gamma in WERI-Rb1. However, neither inducibility of DRA nor DRB mRNA was resulted in an RB-positive stable transformant of WERI-Rb1, WLRB-8. While guanylate-binding protein (GBP) inducibility indicated that the basic IFN-gamma signal transduction pathway remained intact in WERI-Rb1, mRNA for class II transactivator (CIITA), a mediator of the IFN-gamma activation of the HLA class II genes and several other genes related to immune function, was not detectable in IFN-gamma-treated WERI-Rb1, indicating that the lack of CIITA expression was responsible, at least in part, for the inability of RB to rescue HLA class II-inducibility. The HLA class II-associated invariant chain (Ii), the expression of which is also up-regulated by CIITA, was non-inducible in WERI-Rb1, consistent with non-inducible CIITA. Also, IFN-gamma failed to activate the DRA, DRB and Ii promoters in WERI-Rb1. However, exogenous CIITA expression in WERI-Rb1 activated the DRA, DRB and Ii promoter-chloramphinocol acetyltransferase constructs, confirming that CIITA was not induced in WERI-Rb1 and indicating that other proteins required for activation of the class II and Ii promoters were functional in this cell line. Examination of additional cell lines for GBP and CIITA induction revealed that a specific lack of the CIITA IFN-gamma response is common in human tumour lines. The possible role of CIITA defects in tumorigenesis is discussed.

Chloramphenicol O-Acetyltransferase↗
PubMed1996

Vitamin D3 reduces the apoptotic effect of IFN-gamma but does not facilitate HLA class II inducibility in RB-defective cells.

The retinoblastoma protein (RB) regulates the cell cycle by binding and inactivating the E2F transcription factors, which prevents transcription of genes required for DNA synthesis. RB has been shown to inhibit IFN-gamma-mediated apoptosis, possibly by regulating premature entry into S phase. RB is also required for high level IFN-gamma induction of HLA class II genes, which encode antigen presenting molecules, but not for other IFN-gamma inducible genes as demonstrated in previous reports describing the analysis of RB-transformants of the RB-defective cell lines, MDA-468-S4 (S4) and H2009. The IFN-gamma response of the HLA class II genes takes much longer than does the response of the other IFN-gamma inducible genes, raising the question of whether RB facilitates HLA class II inducibility by maintaining cell viability over the long time course required for HLA class II induction. Thus, we sought to learn whether IFN-gamma induced apoptosis in an RB-defective cell line could be prevented independently of RB and whether doing so would facilitate HLA class II inducibility in the RB-defective line. Our results indicated that cotreating the RB-defective S4 cells with IFN-gamma and Vitamin D3 decreased the number of cells containing subdiploid DNA compared to cells treated with IFN-gamma alone, suggesting that Vitamin D3 reduced IFN-gamma-mediated apoptosis. S4 cells cotreated with Vitamin D3 and IFN-gamma also had decreased cell detachment, further indicating that Vitamin D3 decreased IFN-gamma induced apoptosis. However, Vitamin D3 cotreatment resulted in no detectable increase in HLA-DR, the most prominent HLA class II molecule, indicating that the effect of RB on HLA class II induction is not exclusively due to its ability to inhibit IFN-gamma induced apoptosis.

Apoptosis↗
PubMed1996

Retinoblastoma protein inhibits IFN-gamma induced apoptosis.

Regulation of apoptosis (programmed cell death) is critical for maintaining tissue homeostasis. Recent studies indicate a tight coupling between cellular proliferation and apoptosis as cell cycle regulators such as Cyclin D, E1A and E7 appear to influence both events. Each of these modulators is able to bind to and inhibit the function of the retinoblastoma tumor suppressor protein (RB). RB functions, in part, by binding to and inactivating E2F transcription factors, preventing expression of E2F-activated genes associated with G1/S cell-cycle progression. Loss of functional RB deregulates E2F activity and, depending on cell type and environmental factors, promotes tumorigenesis or apoptotic death. To determine the effect of RB on IFN-gamma induced apoptosis, we treated RB-defective carcinoma cell lines and their respective RB-constituted sister clones with IFN-gamma and examined the cells for alterations characteristic of apoptosis. We observed that RB-defective cells, but not the RB-reconstituted clones, decreased in size following IFN-gamma treatment. IFN-gamma treatment caused increased cell detachment in the RB-defective lines but did not affect adherence of the RB-reconstituted clones. Assays for DNA fragmentation revealed lower molecular weight DNA and the apoptosis-associated oligo-nucleosomal ladder following IFN-gamma treatment of the RB-defective cells while higher molecular weight DNA was present in the IFN-gamma treated, RB-reconstituted lines. IFN gamma-induced apoptosis in RB-defective cells was enhanced by serum stimulation, which is also characteristic of p53-dependent E2F-1-mediated apoptosis. However, IFN-gamma induced apoptosis in RB-defective lines does not require wild-type p53 suggesting that, upon IFN-gamma induction, deregulated E2F-mediated apoptosis can also proceed via p53-independent pathways.

Antineoplastic Agents↗
PubMed1996

Apoptosis-independent retinoblastoma protein rescue of HLA class II messenger RNA IFN-gamma inducibility in non-small cell lung carcinoma cells. Lack of surface class II expression associated with a specific defect in HLA-DRA induction.

Work from our laboratory indicates that HLA class II induction by IFN- gamma in the retinoblastoma (RB) protein-defective breast carcinoma line MDA-468-S4 (S4) requires reconstitution of functional RB. To determine whether RB is required for HLA class 11 expression in multiple tumor types, the RB-defective non-small cell lung carcinoma line H2009 and its RB-reconstituted subclones were examined for class II inducibility. Surface HLA-DR (DR) was not inducible by IFN-gamma in H2009. However, unlike the RB-reconstituted subclones of S4, DR surface expression was not detected in the H2009 RB-positive subclones. IFN-gamma induction of CIITA, a major regulator of class II transcription, suggested that H2009 retained at least part of the IFN-gamma signaling pathway leading to class II expression. Examination of class II mRNA indicated that IFN-gamma induction of RB was rescued in the RB-positive subclones of H2009, confirming the requirement for RB for HLA class II inducibility and revealing that RB is required for inducibility in developmentally distinct tumor types. However, DRA inducibility was not rescued in the H2009 RB-positive subclones, which explained the lack of surface DR induction in the RB-positive H2009 subclones. DPA and DPB were also only weakly inducible in the RB-reconstituted H2009 subclones, compared with the previously described, S4 RB-positive subclones. Finally, data reported here indicates that RB's ability to inhibit IFN-gamma-induced apoptosis is not a viable explanation for why RB expression rescues DRB inducibility in H2009.

Apoptosis↗
PubMed1995

RB and a novel E2F-1 binding protein in MHC class II deficient B-cell lines and normal IFN-gamma induction of the class IL transactivator CIITA in class II non-inducible RB-defective tumor lines.

The major histocompatibility (MHC) class II genes encode cell surface proteins that bind antigenic peptide for presentation to T-cells. The class II proteins are expressed constitutively on B-cells and EBV-transformed B-cells, and are inducible by IFN-gamma on a wide variety of cell types. Retinoblastoma protein (RB) is a tumor suppressor and functions as a transcriptional repressor by binding and inactivating the transactivator E2F-I. RB-defective tumor lines are non-inducible for MHC class II by IFN-gamma, or very weakly inducible, but transfection of 2 different lines with RB expression vectors re-establishes or substantially enhances class II inducibility. Therefore, we examined the RB status of a series of B-cell mutants that are defective in class II expression, generated either in vitro or derived from Bare Lymphocyte Syndrome (BLS) patients. Nuclear matrix-bound RB was detectable in all cases, indicating that loss of RB is not responsible for decreased class II expression in these lines. A second E2F-I binding protein, most likely DP-I, was also apparently normal in both class II-positive and -negative B-cell lines. We also examined the IFN-gamma induction of CIITA in RB-defective lines. CIITA is a class II gene transactivator known to be defective in one form of BLS and to be required for the induction of MHC class II by IFN-gamma. CIITA mRNA is normally inducible by IFN-gamma in class II non-inducible, RB-defective lines, and in one line, re-expression of RB has no effect on CIITA mRNA induction levels. Thus, the block in MHC class II inducibility in RB-defective cells is not due to a block in CIITA inducibility.

Antigens, Nuclear↗