Polymorphisms of p21 cyclin-dependent kinase inhibitor and malignant skin tumors.

Polymorphisms of the cyclin-dependent kinase inhibitor gene p21 have recently been reported to be associated with several human cancers. To determine whether the polymorphisms are also associated with human skin cancers, we investigated the p21 polymorphisms in 165 healthy Japanese and 113 Japanese with malignant skin tumors: 30 squamous cell carcinoma, 20 malignant melanoma, 33 basal cell carcinoma, and 30 Bowen's disease. The p21 polymorphisms were characterized by single base substitutions in the following two sites: the last nucleotide of codon 31 of exon 2 and the site 20 nucleotides downstream from the 3' end of exon 3. The two polymorphic sites were reported to be firmly linked to each other. We have shown that the two sites were firmly linked to each other also in Japanese and that no associations of the polymorphisms with the skin cancers in Japanese were detected by statistical analysis. Although the p21 polymorphisms were found not to be involved with skin carcinogenesis, ethnic differences of the allele frequency distribution must be taken into account in studying the role of the p21 polymorphism in carcinogenesis.

Germ cell-specific expression of green fluorescent protein in transgenic rainbow trout under control of the rainbow trout vasa-like gene promoter.

A technique to identify and isolate live fish primordial germ cells (PGCs) has not been established, in spite of the importance of purified germ cells for molecular and cellular studies. In rainbow trout, the distribution of vasa transcripts is restricted to the germ cell lineage, making this transcript a useful indicator of PGCs. Therefore, in this study, we cloned and characterized the rainbow trout vasa-like gene (RtVLG) regulatory regions and produced transgenic trout carrying the green fluorescent protein (GFP) gene driven by the RtVLG regulatory regions (p vasa-GFP) in order to identify live PGCs in vivo. In transgenic trout carrying the p vasa-GFP construct, cells showing green fluorescence were first observed at the mid-blastula stage; however, no cell-type-specific expression was observed at this stage. At the eyed stage, about 30% of the transgenic embryos showed specific GFP expression in PGCs, and at the hatching stage, about 70% of the transgenic embryos did so. An immunohistochemical study of hatching stage embryos revealed that the GFP-expressing cells are located in genital ridges. This transgenic trout, having visualizable PGCs, will make it possible to isolate live PGCs for in vitro studies and to study the ontogeny of PGCs including sex differentiation in live embryos.

Cloning and characterization of a vasa-like gene in rainbow trout and its expression in the germ cell lineage.

The origin of germ cells and the molecular mechanisms of primordial germ cell (PGC) determination in teleosts are unclear. Vasa is a member of the DEAD protein family and plays an indispensable role in germ cell determination in Drosophila and Xenopus species. In this study, we isolated and characterized a rainbow trout vasa cDNA as a first step towards understanding the molecular mechanisms of PGC determination and development and to develop a molecular marker to identify the PGCs in rainbow trout. Cloning of vasa cDNA was performed by degenerate- and RACE-PCR. The predicted amino acid sequence of rainbow trout Vasa contained eight consensus sequences for the DEAD protein family and five arginine-glycine-glycine repeats, a common character of known Vasa homologues. Overall amino acid similarity to the Vasa of Drosophila was 79.2%. Whole-mount in situ hybridization of eyed stage embryos (eighty somite stage) revealed that signals were localized to the putative PGCs. In adult rainbow trout tissues, both ovaries and testes contained large amounts of vasa gene transcripts. A reverse transcription-polymerase chain reaction analysis of unfertilized eggs proved that trout vasa is a maternal factor. Although we have not determined whether rainbow trout vasa functions as a germ cell determinant, its limited expression in the germ cell lineage proved that rainbow trout vasa can be used as a marker molecule for PGCs. This marker will make it possible to identify the PGCs or presumptive PGCs in early trout embryos whose germ cells can not be distinguished by morphological characteristics.

Structure, expression, and conserved physical linkage of mouse testicular cell adhesion molecule-1 (TCAM-1) gene.

Isolation and characterization were performed for cDNA encoding mouse testicular cell adhesion molecule-1 (TCAM-1) using 2908 bases coding for a protein having 548 amino acids (60 kDa). Mouse TCAM-1 protein was found to consist of seven domains for signal sequence, five immunoglobulin (Ig) domains, and the transmembrane plus cytoplasmic domain. TCAM-1 gene and the region linking it to growth hormone (GH) gene located downstream from the TCAM-1 gene were then analyzed. The mouse TCAM-1 gene was 11.6 kb in length with 8 exons; the same as for the 12.0 kb rat gene. The distance from the TCAM-1 to GH gene was 12.5 kb in the mouse genome, and 7.6 kb in the rat. By Northern hybridization, 3.1-kb TCAM-1 mRNA was detected in 17-day testis and would appear present in pachytene spermatocytes and round spermatids.

p53 gene mutations in squamous cell carcinoma occurring in scars: comparison with p53 protein immunoreactivity.

We determined the relationship between p53 expression and p53 gene mutations in squamous cell carcinoma occurring in scars and unrelated to UV light irradiation. We analyzed biopsy specimens obtained from three patients with squamous cell carcinoma. A monoclonal antibody against p53 (DO-7) was used for the immunohistochemical analysis. p53 gene mutations were detected by the polymerase chain reaction and single-strand conformation polymorphism analysis and direct DNA sequencing. p53 overexpression was observed in atypical squamous cells of one case. Those of two other cases, however, showed negative immunoreactivity to p53. Exon 6 of the p53 gene in all three cases and exon 7 in one case showed electrophoretic mobility shifts in polymerase chain reaction and single-strand conformation polymorphism analysis. DNA sequencing analysis showed a missense mutation and a silent mutation in exon 6 of the case with p53 overexpression, a three-base deletion in exon 6 of one case with no p53 overexpression, and a three-base deletion in exon 6 and a missense mutation in exon 7 of another such case. Although immunohistochemical overexpression of p53 has been thought to result from p53 gene mutations, our results suggest that negative immunoreactivity to p53 also can result from p53 gene mutations, for example, short gene deletions.

Establishment and characterization of an epithelioid sarcoma cell line with an autocrine response to interleukin-6.

A novel epithelioid sarcoma (ES) cell line, designated as ES-OMC-MN, was established from a 44-year-old woman with recurrence and metastasis of ES. The cells were spindle-shaped or polygonal and were positive for cytokeratin and vimentin on immunohistochemical staining. Electron microscopy revealed desmosome-like structures between the cells. These characteristics were also noted in the original tumor. Southern blot analysis of HindIII digests showed an additional 8.0 kb band and an 8.8 kb band in DNA from the cultured cells and the original tumor as well as the peripheral blood cells of the patient, while only an 8.8 kb band was observed in control human placental DNA. There were no point mutations of N-ras codons 12, 13, and 61, suggesting that the abnormality of N-ras was not due to mutation but to polymorphism. Interleukin-6 (IL-6) was secreted into the culture medium at high levels. Recombinant IL-6 augmented the proliferation of these cells, while cell growth was inhibited by incubation with an anti-IL-6 antibody. The cells also expressed surface IL-6 receptors, indicating that IL-6 acted on this cell line in an autocrine manner. IL-6 and IL-6 receptors were also found in the original tumor. These results demonstrate that ES-OMC-MN cells retained all the morphological and biochemical characteristics of the original tumor and suggest that an autocrine effect of IL-6 may be involved in the development of ES.

Metastatic epithelioid sarcoma with an N-ras oncogene mutation.

At age 25 a Japanese woman noticed an elastic-hard nodule 2 cm in diameter on the anterior side of her right leg. The nodule had developed an ulcer in its center. Simple resection was performed several times. However, the lesion recurred repeatedly. The patient underwent amputation of the right leg at the age of 34, because the diagnosis of epithelioid sarcoma was established histologically. No recurrence was observed for 9 years. Recently, the patient noticed multiple painful, ulcerative nodules about 1 cm in diameter on her scalp, trunk, and extremities. She refused extensive resection for a religious reason and died of massive hematemesis. Autopsy revealed metastatic epithelioid sarcoma in the skin, lungs, kidneys, pancreas, transverse colon, thyroid, and sternum. Chromosomal analysis of the tumor revealed various aberrations and an N-ras oncogene mutation.

A statistical study on clinical findings of solar keratosis.

Clinical findings were studied in 50 patients with 53 lesions of solar keratosis encountered during the 15 year period from 1977 to 1991. The majority of these cases were evident in the over-sixty age group (average 62.2 years). A greater proportion were females. Most lesions were observed on the face; especially on the cheek, and from the outer eyelid to the temple. The erythematous type (desquamative-keratotic) lesion was the most evident. With the exceptions of the years 1979 and 1991, no remarkable increase in the number of patients was noted during the 15 year period.

A statistical study on histopathologic findings of solar keratosis.

We examined histopathologic data from 50 patients with 53 lesions of solar keratosis encountered over the past 15 years (1977 to 1991). The epidermis showed acanthosis, uneven epidermal dyeing properties, disorderly arrangement of the basal cells, atypia of the nucleus, mitotic figures and hyperpigmentation of the basal layers. The dermis showed actinic elastosis, cell infiltration mainly consisting of the lymphoid cells, and incontinentia pigmenti histologica. Among histologic types, the hypertrophic type was the primary example.