Altered regulation of MHC class I genes in different tumor cell lines is reflected by distinct sets of DNase I hypersensitive sites.

The capacity of cytotoxic-T lymphocytes to recognize and destroy tumor cells depends on the surface expression by tumor cells of MHC class I molecules loaded with tumor antigen peptides. Loss of MHC-I expression is the most frequent mechanism by which tumor cells evade the immune response. The restoration of MHC-I expression in cancer cells is crucial to enhance their immune destruction, especially in response to cancer immunotherapy. Using mouse models, we recovered MHC-I expression in the MHC-I negative tumor cell lines and analyzed their oncological and immunological profile. Fhit gene transfection induces the restoration of MHC-I expression in highly oncogenic MHC-I-negative murine tumor cell lines and genes of the IFN-γ transduction signal pathway are involved. Fhit-transfected tumor cells proved highly immunogenic, being rejected by a T lymphocyte-mediated immune response. Strikingly, this immune rejection was more frequent in females than in males. The immune response generated protected hosts against the tumor growth of non-transfected cells and against other tumor cells in our murine tumor model. Finally, we also observed a direct correlation between FHIT expression and HLA-I surface expression in human breast tumors. Recovery of Fhit expression on MHC class I negative tumor cells may be a useful immunotherapeutic strategy and may even act as an individualized immunotherapeutic vaccine.

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Further characterization of cis-acting regulatory sequences in the genomic locus of the murine erythropoietin receptor: evidence for stage- specific regulation

Blood ◽

10.1182/blood.v83.5.1428.1428 ◽

1994 ◽

Vol 83 (5) ◽

pp. 1428-1435 ◽

Cited By ~ 11

Author(s):

H Youssoufian

Keyword(s):

Cell Lines ◽

Tissue Specificity ◽

Specific Activity ◽

Dnase I ◽

Erythropoietin Receptor ◽

Erythroid Cell ◽

Regulatory Sequences ◽

Dnase I Hypersensitive Sites ◽

Hypersensitive Sites ◽

Murine Erythroleukemia

Abstract Expression of the murine erythropoietin receptor (EpoR) gene was investigated in progenitor cell lines representing distinct stages of hematopoietic differentiation. In murine erythroid cell lines, the EpoR mRNA level was fivefold higher in the more mature murine erythroleukemia (MEL) cells than in CB-5 cells and very low in granulocyte/macrophage-like FDC-P1 cells. GATA-1 mRNA was present in equivalent levels in both erythroid cell lines, but at a low level in FDC-P1 cells. To account for the elevated levels of EpoR mRNA, the activity of the promoter and expression of DNase I hypersensitive sites were assessed as markers of transcriptional activity in various cell lines. Among a series of 5′ flanking restriction fragments linked to a reporter gene, a 83-bp fragment that includes binding sites for the transcription factors GATA-1 and Sp-1 gave low levels of erythroid- specific activity, and a 256-bp fragment that includes, in addition, two sites for the putative CACCC-binding protein gave the highest level of erythroid-specific transcription. DNase I footprinting showed binding of a constitutive factor to the proximal CACCC-binding site, and deletion or mutation of this site significantly reduced the overall expression while maintaining tissue-specificity. Three DNase I hypersensitive sites were detected in the 5′ flanking region of the EpoR gene, two of which were unique to MEL cells. These sites were situated over the promoter region and approximately 0.5 kb and 2.4 kb upstream of the transcriptional initiation sites. A 0.8-kb restriction fragment spanning the distal site caused approximately a four-fold rise in transcription from the endogenous or a heterologous promoter in MEL cells independent of its orientation and up to 1.5-fold rise in CB-5 cells, but it was inactive in COS-1 cells that were cotransfected with an expression plasmid encoding GATA-1. These results show that (1) basal activity as well as tissue specificity of the EpoR promoter can be accounted for by its interaction with GATA-1, and (2) upstream sites regulate the strength of the promoter. Expression of the distal DNase I hypersensitive site and the corresponding enhancer activity in MEL cells suggests a role for this element in stage-specific transcriptional control.

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HLA class I typing of tumor cell lines using flourescence activated cell sorting (FACS) and complement dependent cytotoxicity (CDC)

Human Immunology ◽

10.1016/0198-8859(93)90480-o ◽

1993 ◽

Vol 37 ◽

pp. 149

Keyword(s):

Tumor Cell ◽

Cell Lines ◽

Cell Sorting ◽

Hla Class I ◽

Class I ◽

Tumor Cell Lines ◽

Complement Dependent Cytotoxicity

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Altered binding of regulatory factors to HLA class I enhancer sequence in human tumor cell lines lacking class I antigen expression.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.89.8.3488 ◽

1992 ◽

Vol 89 (8) ◽

pp. 3488-3492 ◽

Cited By ~ 34

Author(s):

O. Blanchet ◽

J. F. Bourge ◽

H. Zinszner ◽

A. Israel ◽

P. Kourilsky ◽

...

Keyword(s):

Tumor Cell ◽

Cell Lines ◽

Human Tumor ◽

Hla Class I ◽

Antigen Expression ◽

Class I ◽

Tumor Cell Lines ◽

Human Tumor Cell ◽

Human Tumor Cell Lines ◽

Regulatory Factors

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Decreased tumor surveillance in perforin-deficient mice.

Journal of Experimental Medicine ◽

10.1084/jem.184.5.1781 ◽

1996 ◽

Vol 184 (5) ◽

pp. 1781-1790 ◽

Cited By ~ 336

Author(s):

M E van den Broek ◽

D Kägi ◽

F Ossendorp ◽

R Toes ◽

S Vamvakas ◽

...

Keyword(s):

Tumor Cell ◽

Nk Cells ◽

Mhc Class I ◽

Cell Lines ◽

Tumor Cell Lines ◽

Tumor Surveillance ◽

Induced Tumors ◽

Deficient Mice

Immune surveillance against tumors usually depends on T cell recognition of tumor antigens presented by major histocompatibility complex (MHC) molecules, whereas MHC class I- tumors may be controlled by natural killer (NK) cells. Perforin-dependent cytotoxicity is a major effector function of CD8+ MHC class I-restricted T cells and of NK cells. Here, we used perforin-deficient C57BL/6 (PKO) mice to study involvement of perforin and Fas ligand in tumor surveillance in vivo. We induced tumors in PKO and normal C57BL/6 mice by (a) injection of different syngeneic tumor cell lines of different tissue origin in naive and primed mice; (b) administration of the chemical carcinogens methylcholanthrene (MCA) or 12-O-tetradecanoylphorbol-13-acetate (TPA) plus 7,12-dimethylbenzanthracene (DMBA), or (c) by injection of acutely oncogenic Moloney sarcoma virus. The first set of models analyzes the defense against a tumor load given at once, whereas the last two sets give information on immune defense against tumors at the very moment of their generation. Most of the tumor cell lines tested were eliminated 10-100-fold better by C57BL/6 mice in an unprimed situation; after priming, the differences were more pronounced. Lymphoma cells transfected with Fas were controlled 10-fold better by PKO and C57BL/6 mice when compared to untransfected control cells, indicating some role for FasL in tumor control. MCA-induced tumors arose more rapidly and with a higher incidence in PKO mice compared to C57BL/6 or CD8-deficient mice. DMBA+TPA-induced skin papillomas arose with similar high incidence and comparable kinetics in both mouse strains. C57BL/6 and PKO mice have a similar incidence of Moloney murine sarcoma and leukemia virus-induced sarcomas, but tumors are larger and regression is retarded in PKO mice. Thus, perforin-dependent cytotoxicity is not only a crucial mechanism of both cytotoxic T lymphocyte- and NK-dependent resistance to injected tumor cell lines, but also operates during viral and chemical carcinogenesis in vivo. Experiments addressing the role of Fas-dependent cytotoxicity by studying resistance to tumor cell lines that were stably transfected with Fas neither provided evidence for a major role of Fas nor excluded a minor contribution of Fas in tumor surveillance.

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Albumin and alpha-fetoprotein gene transcription in rat hepatoma cell lines is correlated with specific DNA hypomethylation and altered chromatin structure in the 5' region

Molecular and Cellular Biology ◽

10.1128/mcb.7.5.1856-1864.1987 ◽

1987 ◽

Vol 7 (5) ◽

pp. 1856-1864

Author(s):

I Tratner ◽

J L Nahon ◽

J M Sala-Trepat ◽

A Venetianer

Keyword(s):

Cell Lines ◽

Chromatin Structure ◽

Hepatoma Cell ◽

Dnase I ◽

Alpha Fetoprotein ◽

Hepatoma Cell Lines ◽

Dnase I Hypersensitive Sites ◽

Albumin Gene ◽

Hypersensitive Sites ◽

Rat Hepatoma

We examined DNA methylation and DNase I hypersensitivity of the alpha-fetoprotein (AFP) and albumin gene region in hepatoma cell lines which showed drastic differences in the level of expression of these genes. We assayed for methylation of the CCGG sequences by using the restriction enzyme isoschizomers HpaII and MspI. We found two methylation sites located in the 5' region of the AFP gene and one in exon 1 of the albumin gene for which hypomethylation is correlated with gene expression. Another such site, located about 4,000 base pairs upstream from the AFP gene, seems to be correlated with the tissue specificity of the cells. DNase I-hypersensitive sites were mapped by using the indirect end-labeling technique with cloned genomic DNA probes. Three tissue-specific DNase I-hypersensitive sites were mapped in the 5' flanking region of the AFP gene when this gene was transcribed. Similarly, three tissue-specific DNase I-hypersensitive sites were detected upstream from the albumin gene in producing cell lines. In both cases, the most distal sites were maintained after cessation of gene activity and appear to be correlated with the potential expression of the gene. Interestingly, specific methylation sites are localized in the same DNA region as DNase I hypersensitive sites. This suggests that specific alterations of chromatin structure and changes in methylation pattern occur in specific critical regulatory regions upstream from the albumin and AFP genes in rat hepatoma cell lines.

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