MUC1 expression in hematopoietic tissues.

The MUC1 gene encodes the core protein of episialin, which is recognized by several antibodies. Reverse transcription-polymerase chain reaction (RT-PCR) detection of MUC1 transcripts has been proposed for the detection of micrometastases from breast cancers. MUC1 expression in hematopoietic tissues has been reported but not confirmed. Our preliminary RT-PCR studies confirmed MUC1 expression by MDA-231 breast cancer cells. Western blots of MDA-231 proteins stained with anti-MUC1 core gave one 68-kd (core protein) band, with an additional high molecular weight (HMW) band in blots stained with anti-epithelial membrane antigen (EMA). MUC1 expression was detectable by RT-PCR in 4 samples each of peripheral blood, bone marrow, and lymph node. MUC1 expression was detectable by Western blot analysis using anti-MUC1 core and anti-EMA in 2 peripheral blood samples and all bone marrow samples. Western blots from all lymph node samples stained positively with anti-EMA for the HMW product, but the 68-kd product was less prominent. Separated peripheral blood lymphocytes and granulocytes showed similar levels of MUC1 expression. RT-PCR studies demonstrated MUC1 expression in various hematopoietic cell lines. Western blots showed the 68-kd and HMW products in a granulopoietic line, with only the 68-kd product in 3 lymphoblastoid lines. MUC1 is expressed ubiquitously in hematopoietic tissues and is unsuitable for use as a marker for epithelial micrometastases.

Suppression of the tumorigenic phenotype of a rat liver epithelial tumor cell line by the p11.2-p12 region of human chromosome 11.

Comparative chromosomal mapping studies and investigations of tumor-associated chromosomal abnormalities suggest that the development of hepatic tumors in humans and rats may share a common molecular mechanism that involves inactivation of the same tumor suppressor genes or common genetic loci. We investigated the potential of human chromosomes 2 and 11 to suppress the tumorigenic phenotype of rat liver epithelial tumor cell lines. These tumor cell lines (GN6TF and GP7TB) display elevated saturation densities in culture, efficiently form colonies in soft agar, and produce subcutaneous tumors in 100% of syngeneic rat hosts with short latency periods. Introduction of human chromosome 11 by microcell fusion markedly altered the tumorigenicity and the transformed phenotype of GN6TF cells. In contrast, the tumorigenic potential and phenotype of GP7TB cells was unaffected by the introduction of human chromosome 11, indicating that not all rat liver tumor cell lines can be suppressed by loci carried on this chromosome. Introduction of human chromosome 2 had little or no effect on the tumorigenicity or cellular phenotype of either tumor cell line, suggesting the involvement of chromosome 11-specific loci in the suppression of the GN6TF tumor cell line. The GN6TF-11neo microcell hybrid cell lines displayed significantly reduced saturation densities in monolayer cultures, and their ability to grow in soft agar was completely inhibited. Although GN6TF-11neo cells ultimately formed tumors in 80-100% of syngeneic rat hosts, the latency period for tumor formation was much longer. Molecular characterization of GN6TF-11neo microcell hybrid cell lines indicated that some of the clonal lines had spontaneously lost significant portions of the introduced human chromosome, partially delineating the chromosomal location of the putative tumor suppressor locus to the region between the centromere and 11p12. Molecular examination of microcell hybrid-derived tumor cell lines further defined the minimal portion of human chromosome 11 capable of tumor suppression in this model system to the region 11p11.2-p12.

Specific interaction between a Saccharomyces cerevisiae protein and a DNA element associated with certain autonomously replicating sequences.

We have isolated a protein from Saccharomyces cerevisiae that binds specifically to a nucleotide sequence associated with the autonomously replicating sequence (ARS) ARS120, located in the telomeric region of a yeast chromosome. "Footprinting" analysis revealed that a 26-base-pair DNA sequence, 5'-CAAGTGCCGTGCATAATGATGTGGGT-3', was protected by this protein from DNase I digestion. A plasmid containing 48 direct tandem repeats of this oligonucleotide was constructed and used to affinity-purify the binding activity. The purified protein, OBF1 (origin binding factor), showed specific binding to ARS120. The 26-base-pair OBF1-protected sequence was sufficient for the recognition and binding of the protein, since the mobility of a DNA fragment containing the synthetic binding site was retarded in agarose gels when incubated with OBF1. By performing competition experiments with a number of different ARSs, we showed that OBF1 binds tightly to some but not all ARSs. Interestingly, OBF1 does not appear to have a discernible affinity for ARS1 or the ARSs associated with mating type loci, HML alpha and HMRa, which are substrates for a DNA-binding activity reported by others. Since OBF1 appears to bind to DNA associated with a number of ARSs, we suggest that this protein may have a function related to ARS activity, perhaps in the initiation of DNA replication at selected ARSs.