Kazuhiro Asano

RCAS1 antigen is highly expressed in extramammary Paget's disease and in advanced stage squamous cell carcinoma of the skin

Receptor-binding cancer antigen expressed on SiSo cells (RCAS1), which is a type II membrane protein expressed on cervical carcinoma cells, induces apoptosis in RCAS1 receptor expressing cells. RCAS1 is thus presumed to protect tumor cells from immune surveillance by infiltrating RCAS1 receptor-positive immunocytes (Sonoda et al. Int J Oncol 1995; 6: 1899-1904; Nakashima et al. Nature Med 1999; 5: 938-942). We performed immunohistochemical analysis of RCAS1 expression in various skin tumors. RCAS1 was not detected in normal human epidermis. One of 21 seborrheic keratosis (4.8%), one of 12 actinic keratosis (8.3%), two of 16 keratoacanthomas (12.5%), and two of 14 basal cell carcinomas (14.2%) expressed RCAS1. RCAS1 was not detected in Bowens disease (0/17). RCAS1 was positive in 45 of 61 (73.8%) squamous cell carcinomas. Interestingly, the expression of RCAS1 was mostly correlated with clinical stages of squamous cell carcinoma. It was found that 46.1% of stage I, 61.1% of stage II, 85.7% of stage III, and 83.3% of stage IV squamous cell carcinomas were RCAS1-positive. In addition, RCAS1 was found to be highly expressed in extramammary Pagets disease. Fifty nine of 63 extramammary Pagets disease samples (93.7%) were positive for RCAS1. Fifty eight (92%) showed co-expression of RCAS1 and carcinoembryonic antigen (CEA). While two of 24 cases of melanoma (8.3%) expressed RCAS1 antigen, none of 20 cases of nevus pigmentosus showed positive staining. These results indicate that RCAS1 is a highly sensitive marker for extramammary Pagets disease. RCAS1 is also expressed in various skin tumors including squamous cell carcinoma, where positive correlation with clinical staging was documented.

Cornified cell envelope formation is distinct from apoptosis in epidermal keratinocytes.

In order to maintain epidermal structural homeostasis, keratinocytes need to modulate their proliferation, differentiation, and cell death. Although terminal differentiation of keratinocytes is characterized by cornified cell envelope (CE) formation and one major mechanism of cell death is apoptosis, the precise relationship between these processes remains obscure. Using normal human cultured keratinocytes (NHK), we compared A23187-induced CE formation and ultraviolet B irradiation (UVB)-induced apoptosis. A23187 stimulated CE formation in 1 mM Ca(2+)-pretreated NHK cells. CE formation was detected by 1 h and the maximal induction was observed at 6 h. Morphological analysis using acridine orange staining revealed that UVB-irradiated NHK cells show distinctive round, homogeneous fragmented nuclear beads, a characteristic feature of apoptotic cells, while A23187-treated cells showed enlarged nuclei with weak chromatin staining, which is not typical of apoptosis. The UVB-irradiated NHK cells did not show CE formation. Caspase activation is a characteristic event during apoptosis. Although UVB irradiation increased caspase 3 activity, no increase in caspase 3 activity was detected during A23187-induced CE formation. Multiple nucleosome-sized fragments of DNA were observed in UVB-treated NHK cells, but not in A23187-treated NHK cells. FACS analyses using anti-annexin V antibody and propidium iodide (PI) showed that UVB irradiation induced both annexin V-positive and PI-negative early apoptotic cells and annexin V-positive and PI-positive late apoptotic cells. On the other hand, A23187-treated NHK cells showed only annexin V-negative and PI-positive non-apoptotic dying cells. Cell death assay revealed a significantly increased apoptotic cells in UVB-irradiated NHK cells, but not in A23187-treated NHK cells. UVB irradiated NHK cells showed increased cytosolic transglutaminase activity, while A23187-treated NHK cells showed increased membrane-associated transglutaminase activity. These results indicate that CE formation is distinct from apoptosis in epidermal keratinocytes.

Structure and Transcriptional Regulation of the Human Cystatin A Gene THE 12-O-TETRADECANOYLPHORBOL-13-ACETATE (TPA) RESPONSIVE ELEMENT-2 SITE (−272 TO −278) ON CYSTATIN A GENE IS CRITICAL FOR TPA-DEPENDENT REGULATION

Cystatin A, a cysteine proteinase inhibitor, is one of the precursor proteins of cornified cell envelope of keratinocytes and is expressed during the late stage of keratinocyte differentiation. We have isolated and characterized the human cystatin A gene. The cystatin A gene consists of three exons and two introns. The first, the second, and the third exons consist of coding sequences that are 66, 102, and 126 base pairs in length, respectively. The first and the second introns consist of 14 and 3.6 kilobase pairs, respectively. The transcription initiation site was located 55 base pairs upstream from the first translation site. The fragment, +77 to −2595 in the 5′-flanking region of the human cystatin A gene, was subcloned into a chloramphenicol acetyltransferase (CAT) reporter vector. The expression vector, p2672CAT, produced a significant CAT activity in transiently transfected SV40-transformed human keratinocytes (SVHK cells), that were further stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent protein kinase C activator. Sequence analysis of the gene detected three TPA responsive elements (TRE-1, TRE-2, and TRE-3) and one AP-2 site on the 5′ upstream promoter region. Deletion analyses of the p2672CAT vector demonstrated that TRE-2, which was located between −272 and −278, was critical for the regulation by TPA. Gel shift analyses revealed that c-Jun, JunD, and c-Fos bound to the TRE-2 region and that the p2672CAT activity level was elevated by co-transfection with c-Jun and c-Fos or with JunD and c-Fos expression vectors. Furthermore, co-transfection of SVHK cells with the protein kinase C-α expression vector and the p2672CAT expression vector also resulted in an increased CAT activity. These results indicate that the 5′-flanking region of the human cystatin A gene confers promoter activity and contains a TRE (TRE-2) that mediates, at least in part, the enhanced expression of this gene by TPA.

Desensitization of the epidermal adenylate cyclase system: agonists and phorbol esters desensitize by independent mechanisms

Exposure of pig epidermis to adenylate cyclase stimulators results in receptor-specific desensitization. We investigated the nature of the agonist-induced desensitization, which was compared with the phorbol ester-induced, receptor-nonspecific desensitization. Both phorbol ester-induced desensitization and the agonist-induced desensitization were accompanied by an increase in forskolin- and cholera toxin-induced cyclic AMP accumulations. The magnitude of the increase in the agonist-induced desensitization was parallel to the degree of the initial cyclic AMP accumulation; histamine and adenosine, which increase more cyclic AMP than epinephrine, resulted in a more marked increase in forskolin- and cholera toxin-induced cyclic AMP accumulations. Similarly, epidermis desensitized to multiple receptors revealed more marked forskolin- and cholera toxin-induced cyclic AMP accumulations than epidermis desensitized to a single receptor. In contrast to the phorbol ester-induced desensitization, agonist-induced desensitization was not affected by the protein kinase C inhibitors H-7 and staurosporin. Further, agonist-induced desensitization was still inducible in phorbol ester-desensitized epidermis and vice versa. In contrast to the agonist-induced desensitization, which is accompanied by the preceding adenylate cyclase stimulation, no evidence for the stimulation of the adenylate cyclase during phorbol ester treatment was obtained. Neither agonist-induced desensitization nor phorbol ester-induced desensitization affected the content of inhibitory guanine nucleotide binding protein of the epidermis, which was monitored by the pertussis toxin (IAP)-catalyzed ADP ribosylation reaction. Our results indicate that agonist-induced desensitization and the phorbol ester-induced desensitization are independent of each other. Although both processes are characterized by increased forskolin- and toxin-induced cyclic AMP accumulations, the former is accompanied by initial cyclic AMP accumulation; the latter is not.

A case of penile caroinoma with lung metastasis.

We report a case of penile carcinoma of a 67 years old man. A 2cm-sized gradually enlarging penile ulcerative tumor was observed, that was accompanied by scrotal swelling. Histopathology revealed well differentiated squamous cell carcinoma with focal detection of human papilloma virus antigen. The patient was assessed to be clinical stage IV of UICC-TNM classification. Following chemotherapy (BLM 30mg, MTX 70mg, CDDP 50mg), total penectomy was performed and tissue defect was covered with scrotal skin. Bilateral regional lymphadenectomy disclosed two metastatic left inguinal lymph nodes. The patient showed no evidence for local recurrence, but died for lung metastasis. [Skin Cancer (Japan) 2000; 15: 61-65]

Successful treatment of primary cutaneous B cell lymphoma with total skin electron beem therapy.

A case of cutaneous B cell lymphoma is reported. The patient was a 82-year-old female, who developed multiple indurated plaques and subcutaneous nodules on her trunk during past 6 months. Histopathology of the skin lesion showed diffuse infiltrate of atypical mononuclear cells, that were positive for CD19, CD20, CD10, CD79a, and HLA-DR. Surface immunoglobulin was negative. Gene analyses disclosed that the tumor cells were positive for immunoglobulin gene and T cell receptor δ1 gene rearrangements. We tried total skin electron beam therapy, that resulted in a complete remission of the skin lesion. [Skin Cancer (Japan) 2000; 15: 71-74]

A case of Amelanotic melanoma.

A 40-year-old woman visited our hospital because of a dome-shaped light red eroded-surfaced tumor measured 1.5×1.9×1.0cm on her right heel. On closer examination, a small dark brown pigmented macule was observed in its peripherary. Excision of the tumor disclosed the tumor to be amelanotic melanoma. The tumor was composed of anaplastic epithelioid cell nests with junctional activity. No melanin granules were appreciated. The tumor cells were positive for S100 and HMB45. Two weeks later wide local excision, sentinel node biopsy and ilio-inguinal lymph node dissection were performed. Although her right inguinal nodes were clinically palpable, no evidence for lymph node metastasis was obtained. The patient was diagnosed as amelanotic melanoma stage IIIA (pT4aN0M0 tumor thickness 4.4mm) and is currently followed without evidance for metastasis. This is the 3rd amelanotic melanoma patient among 91 cases malignant melanoma (3.3%) in our department. [Skin Cancer (Japan) 2000; 15: 185-188]