The decreased susceptibility of metastatic melanoma cells to killing involves an alteration of CTL reactivity.

Metastases are known to be more resistant to therapy than matching primary tumors, in particular they are less prone to apoptosis. In this study we investigated the functional interaction of a CTL clone (LT12) specific for a melanoma TA with the primary tumor (T1) versus its metastatic counterpart (G1). The CTL clone (LT12) was shown to lyse the primary T1 cells more efficiently in a classical cytotoxicity test.

Prion protein prevents human breast carcinoma cell line from tumor necrosis factor alpha-induced cell death.

To define genetic determinants of tumor cell resistance to the cytotoxic action of tumor necrosis factor alpha (TNF), we have applied cDNA microarrays to a human breast carcinoma TNF-sensitive MCF7 cell line and its established TNF-resistant clone. Of a total of 5760 samples of cDNA examined, 3.6% were found to be differentially expressed in TNF-resistant 1001 cells as compared with TNF-sensitive MCF7 cells.

[Tumor/cytotoxic effector cross-talk in the control of tumor susceptibility to lysis].

During the two least decades, the field of tumor immunology has met an expansion of knowledge about the molecular and cellular bases of immune regulation. The identification of cancer antigens has been of critical importance and cancer vaccine is at present a very fast moving field. However, the immunotherapy approaches in cancer are of modest success.

Inhibition of tumor growth and metastatic spreading by overexpression of inter-alpha-trypsin inhibitor family chains.

The inter-alpha trypsin inhibitor (ITI) family is a group of proteins built up from different combinations of I light chain (ITI-L) and 3 highly homologous heavy chains (ITI-HI, -H2 and -H3). To investigate a potential role of the ITI family chains in cancer and metastasis spreading, we engineered human H460M cell lines expressing both the green fluorescent protein (GFP) and one of these chains. These clones were subcutaneously injected in athymic nude mice, and lung metastasis number and primary tumor weight were determined after 28 days.

A model of spontaneous lung metastases visualised in fresh host tissue by green fluorescent protein expression.

The authors describe a model of spontaneous lung metastases in nude mice using green fluorescent protein (GFP) expression as a marker. The human lung cell line H460M was transfected with the humanised GFP-S65T cDNA and a stable fluorescent cell line termed H460M(GFP) was obtained. The latter kept in vitro biological features when compared to the parental H460M cell line, which suggests that GFP-expression does not influence H460M(GFP) cell line behaviour.

Immunohistochemical distribution of inter-alpha-trypsin inhibitor chains in normal and malignant human lung tissue.

The inter-alpha-trypsin inhibitor (ITI) family is a group of plasma proteins built up from heavy (HC1, HC2, HC3) and light (bikunin) chains synthesized in the liver. In this study we determined the distribution of ITI constitutive chains in normal and cancerous lung tissues using polyclonal antibodies. In normal lung tissue, H2, H3, and bikunin chains were found in polymorphonuclear cells, whereas H1 and bikunin proteins were found in mast cells.

Assembly and secretion of recombinant chains of human inter-alpha-trypsin inhibitor in COS-7 cells.

The inter-alpha-trypsin inhibitor (ITI) family is a group of structurally related plasma serine protease inhibitors. The ITI family members consist of combinations of mature heavy chains named HC1, HC2, HC3 linked to bikunin (a Kunitz-type protease inhibitor) by a covalent interchain protein-glycosaminoglycan-protein cross-link. The biosynthesis of the ITI family members takes place in the liver.

Human inter-alpha-trypsin inhibitor heavy chain H3 gene. Genomic organization, promoter analysis, and gene linkage.

To understand more about the human inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3) expression and the relationship between this gene and the family of other ITI heavy chain genes, an analysis of the structure of the ITIH3 gene and its promoter region was performed. This gene is a single copy gene, 14 kilobase pair in length and consists of 22 exons. ITIH3 shares highly conserved exon size and intron-exon borders with other ITI heavy chain genes.

Transverse formamide gradients as a simple and easy way to optimise DNA single-strand conformation polymorphism analysis.

Although widely used, the detection of DNA mutations by the single-strand conformation polymorphism (SSCP) method is often hampered by the need to examine a large set of electrophoretic conditions in order to select the one suited to the DNA sequence under study. We show here that the use of transverse chemical gradient gels allows for a quick and easy optimisation of SSCP analysis, as exemplified on two mutations in exon 2 of the alpha-1-antitrypsin gene.

Inter-alpha-trypsin inhibitor proteoglycan family--a group of proteins binding and stabilizing the extracellular matrix.

Extracellular matrix (ECM) is composed of several macromolecules associated in a complex network. This structure allows cells to adhere, migrate and interact. Hyaluronic acid (HA) is a glycosaminoglycan (GAG) and a major representative of ECM.

Frequency and prognostic evaluation of 3p21-22 allelic losses in non-small-cell lung cancer.

Previous loss of heterozygosity (LOH) studies of chromosome 3p loci have displayed a 60% deletion frequency in non-small-cell lung cancers (NSCLC), as opposed to small-cell lung cancers, in which the 3p deletion is consistently found. However, the high stromal-cell admixture found in NSCLC and the use of the Southern-blot method lead to under-evaluation of this frequency. In this study, we used a very precise microdissection technique followed by PCR amplification of 6 3p21-22 polymorphic genomic sequences to analyze LOH in 86 NSCLC and in normal adjacent tissue.

Involvement of the three inter-alpha-trypsin inhibitor (ITI) heavy chains in each member of the serum ITI family.

Partial cDNAs coding for each of the three human inter-alpha-trypsin inhibitor (ITI) heavy chains were expressed in a bacterial plasmid system and rabbits were immunised with the fusion peptides obtained. Despite the strong sequence homology of these chains, the antisera turned out to be highly specific in the analysis of corresponding mRNA translation products or partially digested serum ITI. Besides classical serum ITI members, their use in Western blotting made it possible to evidence an H3-related ITI form and a low-amount H1-related HC/bikunin component.

Gene expression and protein distribution of inter-alpha-trypsin inhibitor in three human hepatoma cell lines.

In standard culture conditions, three human hepatoma cell lines, Hep3B, PLC/PRF/5 and HepG2, were characterised by a predominant transcription of only two (H2 and L) among the four genes involved in the synthesis of inter-alpha-trypsin inhibitor (ITI)-related proteins. Pulse-chase experiments followed by immuno-precipitation with specific anti-L and anti-H ITI antisera showed that the proteins synthesised displayed a restricted L and/or H2 antigenic reactivity. Furthermore, while Hep3B and PLC/PRF/5 lines only synthesised ITI precursors (mainly the L-form), HepG2 cells were able to secrete an ITI-like protein.

The human inter-alpha-trypsin inhibitor genes respond differently to interleukin-6 in HepG2 cells.

The effects of interleukin 6 (IL-6), the major inducer of the acute-phase reaction, on the expression of inter-alpha-trypsin inhibitor (ITI) genes were examined using human HepG2 hepatoma cells. The three ITI heavy-chain genes H1, H2 and H3 were transcriptionally regulated by IL-6 in a dose- and time-dependent manner. The treatment of HepG2 cells with IL-6 resulted in an increase of H1 and H3 mRNA levels and a decrease of H2 and L mRNA levels.

Tandem orientation of the inter-alpha-trypsin inhibitor heavy chain H1 and H3 genes.

The inter-alpha-trypsin inhibitor H1 (ITIH1) and inter-alpha-trypsin inhibitor H3 (ITIH3) genes have both previously been mapped to chromosomes 3 and 14 in the human and mouse, respectively. We now present evidence that these genes are physically linked. By using cDNA probes, a recombinant DNA phage has been isolated from a bacteriophage DNA library, which contains sequences flanking the 5' end of the ITIH3 gene and the 3' end of the ITIH1 gene.

Post-translational processing of the inter-alpha-trypsin inhibitor in the human hepatoma HepG2 cell line.

In human hepatoma HepG2 cells, the serum inter-alpha-trypsin inhibitor (ITI)-like protein is synthesized from two protein precursors, the heavy chain (H) H2 and the light chain (L). Both of them carry sulphate groups involved in the chondroitin sulphate glycosaminoglycan (GAG) linkage, as demonstrated by [35S]sulphate labelling, chondroitinase digestion and inhibition with beta-D-xyloside, an artificial GAG acceptor. While inhibition of N-glycosylation prevented neither the maturation nor the secretion of the ITI-related entities, brefeldin A induced the accumulation of H and L precursors in the cells, therefore blocking subsequent association and maturation of the precursors before their secretion.

The polymorphism of the plasma inter-alpha-trypsin inhibitor (ITI) and its relationship to the heavy chain H1 subunit gene (ITIH1) at 3p211-212.

We investigated the ITI protein polymorphism in linkage analysis, using DraI and SstI as restriction fragment length polymorphism (RFLP) markers for the ITIH1 gene. Isoelectric focusing (IEF) classification from 76 individual plasma samples and RFLP analysis from the corresponding DNA preparations disclosed linkage disequilibrium between the phenotypic IEF patterns of the two common ITI alleles, ITI*1 and ITI*2, and the diallelic DNA polymorphisms of two ITIH1 RFLPs, represented by DraI 4.0 kb and DraI 2.

Light microscopical detection of inter-alpha-trypsin inhibitor and its different mRNAs in cultured hepatoma Hep G2 cells using immunocytochemical and in situ hybridization techniques.

The Hep G2 hepatoma cell line synthesizes the inter-alpha-trypsin inhibitor (ITI). This protease inhibitor and the other proteins of this family include four polypeptides chains: three heavy chains (HC1, HC2, HC3) and one light chain (bikunin). In the present study, we have demonstrated by immunofluorescence that ITI is detected mainly in perinuclear cytoplasmic zones comparable to those of albumin or alpha-1-antitrypsin.

Detection by the polymerase chain reaction of two polymorphisms in exon 14 of the human inter-alpha-trypsin inhibitor heavy chain H1 gene.

Two polymorphisms were detected within exon 14 of the inter-alpha-trypsin inhibitor heavy chain H1 (ITIH1) gene. The polymorphisms are detected by digesting the same 202-bp polymerase reaction product with the PstI and Hph1 restriction endonucleases. These gene polymorphisms lead to the change of two amino acids in the mature protein.

Isolation and characterization of the human inter-alpha-trypsin inhibitor heavy-chain H1 gene.

The human inter-alpha-trypsin inhibitor heavy chain H1 (ITI heavy chain H1) gene was isolated from two overlapping clones. It spans 14 kbp and is composed of 22 exons from 15 bp to 281 bp in size and has consensus splice sites. Intron sizes range from 80 bp to 2000 bp.

Human pre-alpha-trypsin inhibitor-precursor heavy chain. cDNA and deduced amino-acid sequence.

Pre-alpha-trypsin inhibitor (P alpha I) is a serine-proteinase inhibitor of M(r) 130,000 found in human serum. This protein belongs to the family of proteins called inter-alpha-trypsin inhibitor (ITI). P alpha I is composed of a heavy chain (HC3) and of a light chain (bikunin), synthesized by two separate mRNA.

Human inter-alpha-trypsin inhibitor: full-length cDNA sequence of the heavy chain H1.

Inter-alpha-trypsin inhibitor (ITI), called inter-alpha-inhibitor, is a 220 kDa serine proteinase inhibitor found in human serum. It is composed of at least three distinct polypeptide chains. These chains, named H1, H2 and L, are an independently synthesized and proteolytically processed precursor protein.

The genes for the inter-alpha-inhibitor family share a homologous organization in human and mouse.

Inter-alpha-inhibitor (I alpha I) and related molecules in human are comprised of three evolutionarily related, heavy (H) chains and one light (L) chain, also termed bikunin. The latter originates from a precursor molecule that is cleaved to yield the bikunin and another protein designated alpha-1-microglobulin (A1m). The four H and L chains are encoded by four distinct genes designated H1, H2, H3, and L.

Structural analysis of the human inter-alpha-trypsin inhibitor light-chain gene.

The human inter-alpha-trypsin inhibitor (ITI) light-chain gene, which codes for the two proteins alpha 1-microglobulin (protein HC) and ITI-derived human inhibitor of 30 kDa (HI-30), was isolated from a human genomic library. This gene, present as a single copy in the human genome, is composed of 10 exons and 9 introns distributed over 20 kbp. A single transcriptional initiation site was identified in the 5'-flanking region which contained promoter elements, but no typical TATA box.