Altered surface expression and increased turnover of the alpha6beta4 integrin in an undifferentiated carcinoma.

The integrin alpha6beta4, predominantly expressed on tissues of epithelial origin, is known to be variably expressed on carcinomas. The biochemical changes resulting in altered expression during tumor progression are unknown. We have analyzed the expression of alpha6beta4 in a multi-step mouse model of skin carcinogenesis representing normal keratinocyte, benign papilloma and malignant undifferentiated carcinoma. All cell lines expressed the alpha6 integrin exclusively as the alpha6beta4 integrin heterodimer. Analysis of this integrin by flow cytometry and immunoprecipitation of surface labeled proteins revealed that the undifferentiated carcinoma cells have an approximately 75% reduction in surface expression of the integrin as compared with the keratinocyte and papilloma cell lines. The alpha6beta4 integrin which remains expressed on the carcinoma cells is diffusely distributed in the membrane and has an approximately 2.5-fold increased biological turnover as compared with normal keratinocytes. The decreased biological half-life and the loss of polarized expression of alpha6beta4 on the carcinoma cells suggests an altered functional role for the alpha6beta4 integrin on carcinoma cells during tumor progression. These factors may contribute to the known supression of hemidesmosome structures and the increased migration phenotype associated with some epithelial carcinomas.

Characterization of integrin subunits, cellular adhesion and tumorgenicity of four human prostate cell lines.

Cellular adhesion to extracellular matrix proteins via integrin molecules is a major factor in the process of invasion and metastasis of human tumor cells. Four human prostate cell lines were characterized according to the presence and quantity of integrin subunits, the ability of the cells to attach to extracellular substrates and the capacity of the cells to form tumors in severe combined immunodeficient (SCID) mice. All four human prostate cell lines expressed three to five integrins on their cell surfaces. The DU145, PC3 and 431P cells expressed primarily alpha 3, alpha 5, and alpha 6 integrin at similar levels. These cell lines expressed the subunits beta 1, beta 3, and beta 4 with beta 1 predominant. The DU145 cells preferred attachment to fibronectin, followed by laminin and vitronectin. Approximately 50%-60% of the binding of DU145 cells to fibronectin and laminin was dependent on the function of alpha 5 beta 1 and alpha 6 respectively. The cell line LNCaP differed in its low expression of the alpha 3 subunit, 95% of cellular adhesion to fibronectin and laminin being integrin-dependent and its inability to attach to vitronectin, in spite of surface expression of alpha v beta 3. All the cell lines except for LNCaP readily formed tumors within SCID mice and the expression of alpha 3, alpha 6, beta 1 and beta 4 integrin subunits was preserved in the resulting tumor tissue. The altered adhesion properties of the LNCaP cells may explain their altered tumorigenicity.

Development and utilization of a somatic cell hybrid mapping panel to assign NotI linking probes to the long arm of human chromosome 6.

A somatic cell hybrid mapping panel that defines seven regions of the long arm and one region of the short arm of human chromosome 6 has been developed. Utilizing this panel, 17 NotI boundary clones from a NotI linking library were regionally assigned to the long arm of chromosome 6. The majority of these clones (11) were found to localize within band regions 6q24-q27. The nonuniform distribution of NotI sites may indicate a cluster of HTF islands and likely represents a coincidence of coding sequences in this region of chromosome 6. Cross-hybridization of these linking clones to DNA from other species (zoo blots) provides further evidence for transcribed sequences in 7 of the NotI clones. These NotI clones were also used to identify corresponding NotI fragments using pulsed-field gel electrophoresis, facilitating further physical mapping of this region. Finally, regional assignment of five polymorphic probes to the long arm of chromosome 6 is also presented. These hybrids and probes should facilitate the construction of a physical and genetic linkage map to assist in the identification of disease loci along chromosome 6.

Tumorigenicity in human melanoma cell lines controlled by introduction of human chromosome 6.

Chromosome banding analysis of human malignant melanoma has documented the nonrandom alteration of chromosome 6. To determine the relevance of chromosome 6 abnormalities in melanoma, a normal chromosome 6 was directly introduced into melanoma cell lines. The resulting (+6) microcell hybrids were significantly altered in their phenotypic properties in culture and lost their ability to form tumors in nude mice. The loss of the chromosome 6 from melanoma microcell hybrids resulted in the reversion to tumorigenicity of these cells in mice. The introduction of the selectable marker (psv2neo) alone into melanoma cell lines had no effect on tumorigenicity. These results support the idea that one or more genes on chromosome 6 may control the malignant expression of human melanoma.

Molecular mapping of the oncogene MYB and rearrangements in malignant melanoma.

The human cellular oncogene MYB has been mapped to 6q22-q23. Deletions and translocations involving this region of the long arm of chromosome 6 occur frequently in human malignant melanoma, and there are anecdotal reports of MYB gene rearrangements in this cancer. In the current study, Southern blotting and pulsed field gel electrophoresis (PFGE) have been performed to determine whether MYB or its flanking regions are commonly altered in malignant melanoma. Southern blotting failed to document obvious rearrangement of the MYB gene in 15 cases studied. To extend analysis of the MYB region, a long-range restriction map was established by PFGE. This map was then linked to the known restriction map of frequent cutting enzymes. Based on the mapping data and analysis of the MYB region in melanomas, ClaI tissue-specific variation due to methylation was demonstrated. Also, two melanomas (containing alterations in band 6q13) also demonstrated by PFGE a unique restriction fragment for the MYB gene. These results extend significantly the physical map surrounding the MYB locus and provide further evidence for the rearrangement of chromosome 6 in malignant melanoma.