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Keratin (K) expression index has been reported to be related to cell invasion activity in adenocarcinoma. In a previous study, we observed a negative correlation between K expression and cell invasion activity; i.e., when many Ks are expressed in the cells, the cell activity is low. To further elucidate the correlation between Ks and invasion activity, RNA interference experiments of K8, K18 and K19 were carried out to clarify the essential role of Ks using T24 and HEC-1 as typical squamous cell carcinoma and adenocarcinoma cells, respectively. K8 small interfering RNA (siRNA) was most effective against K18 and K19 expression and demonstrated the strongest effect on relative invasion activity among the siRNAs used. These results suggest that K8/K18 or K8/K19 filaments may play roles in internal cell structure and invasion activity. Moreover, K18 and K19 were capable of substituting for each other, and K18 or K19 formed filaments with K8. In addition, cells treated with K8 siRNA demonstrated high invasion activity, which was approximately double that observed with control siRNA in HEC-1 cells. The order of effects was K8>K19>K18 in the two cell lines. The above results suggest that K8 may play a signifiant role in invasive functions in epithelial and metastatic cells.

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Hybrid cyst is a rare cystic lesion that includes more than two components of the pilosebaceous units. To clarify the clinical and pathological features of hybrid cysts, we report two cases and review 15 cases of hybrid cyst in Japan. On the whole, the age range was 12-73 years with a 2.95:1 female predominance and predilection for the scalp and face (46.7%). Most of the tumours presented as a solitary lesion and the size range was 2-45 mm. The most frequent histological type was the combination of infundibular and trichilemmal cysts (60.0%). Studying the clinicopathological features of hybrid cysts helps us in understanding the pathogenesis of diseases arising from pilosebaceous units.

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BACKGROUND: Angiotensin receptors are the specific receptors of angiotensin II of the renin-angiotensin system. However, expression of the receptors in hair follicles has not been determined. OBJECTS: To clarify the expression and localization of angiotensin receptors in human anagen hair follicles and basal cell carcinomas. METHODS: We studied immunohistochemically the expression of angiotensin type 1(AT1) and type 2 (AT2) receptors in human anagen hair follicles and in 16 cases of basal cell carcinoma (BCC) (nine of solid BCC of the circumscribed type, two of adenoid BCC, five of BCC with follicular differentiation). RESULTS: Our experiments demonstrated the localization of AT1 in the inner root sheath and the inner layers of the outer root sheath. In BCC, positive staining with AT1 was revealed in the tumour cells of basal cell carcinoma with follicular differentiation. CONCLUSIONS: AT1 may have a role in association with follicular keratinization. Studying AT1 distribution may be useful in understanding the pathophysiology of human hair follicles and the hair follicle-associated tumours.

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BACKGROUND: Previous studies have demonstrated that synthetic cell-permeable analogues of ceramide promote differentiation and inhibit proliferation of keratinocytes, and that the vitamin D3 inducible sphingomyelin cycle generates ceramide in keratinocytes. Although it has been suggested that exogenous ceramide induces apoptosis of keratinocytes, which is similar to their effect on other cell types, such as leukaemia cells, only a few studies have reported ceramide-induced apoptosis of keratinocytes. OBJECTIVE: To determine whether ceramide induces apoptosis of keratinocytes, we used the synthetic ceramide analogue, C2-ceramide (N-acetylsphingosine) and a human squamous cell carcinoma cell line, HSC-I. METHODS: We treated HSC-I cells with C2-ceramide, followed by a viability assay, morphological observations, nick end-labelling (TUNEL), DNA electrophoresis, and electron microscopy. RESULTS: In the viability assay, C2-ceramide was toxic to HSC-I cells in a dose-dependent manner. Manifestations of apoptotic morphology occurred in the ceramide-treated cells, whereas these morphological changes did not occur in cells treated with dihydroceramide (N-acetylsphinganine). TUNEL revealed that many of the ceramide-treated cells showed positive reactivity. DNA electrophoresis demonstrated that C2-ceramide caused internucleosomal fragmentation in a dose- and time-dependent manner. Electron microscopy revealed that the ceramide-treated cells manifested morphological characteristics typical of apoptosis. CONCLUSIONS: The present results demonstrate that C2-ceramide induces apoptosis of transformed human keratinocytes, whereas C2-dihydroceramide does not have such an effect. The fact that ceramide induces apoptosis of keratinocyctes raises the possibility that intracellular ceramide, which is increased with differentiation of the epidermis, might be involved in terminal differentiation, a specialized form of apoptosis of keratinocytes.

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The synthesis of keratin is considered to occur in epithelial and epidermal cells. Previous studies have not reported on keratin synthesis within melanocytes that derive from neural crest cells. Epithelial and neural crest cells originally develop from ectodermal tissue. We previously reported that the expression of keratin is a universal phenomenon seen in cultured melanoma cell lines, as demonstrated by two-dimensional polyacrylamide gel electrophoresis, western blot, and electron microscopy analyses. To further investigate the specificity of keratin function in melanocytic cells, we first examined the presence of keratin proteins in cultured human melanocytes, and unexpectedly found keratin subunits in melanocytes by the above-mentioned procedures. The keratin (K) subunits were composed of K1, K5, K8, K10, K14, K16, and K18, together with vimentin. Neural crest cells, which contain immature embryonic melanocytes developing from ectoderm, already expressed keratins; however, under electron microscopy, the expressed keratin did not form filamentous structures. Although the ATP synthase alpha-chain, which is expressed universally in cultured epidermal tumor cell lines, was also expressed in cultured melanocytes and neural crest cells, a novel malignant melanoma-related protein (MMRP) was absent in melanocytes and neural crest cells. We concluded that keratin subunits are present in both cells, but do not construct keratin filaments.

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Keratin is an intermediate filament that is a major structural protein of epithelial cells. Until now, the expression of keratin in melanoma cells has not been well understood. Recently, it has been reported that keratin expression is correlated with invasive and metastatic behavior in a variety of cell types. We report keratin expression in cultured murine melanoma cell lines B16-F1 (low incidence of lung colonization) and F10 (high incidence of lung colonization) using an aqueous solution (10 mM Tris-HCl (pH 7.4)/10 mM EDTA/phenylmethyl sulphonyl fluoride (PMSF, 10 micrograms/ml). By comparing these two cell lines, we investigated whether differences in keratin expression can influence the metastatic ability of tumor cell lines in vitro. However, no remarkable differences in keratin expression were found in these cell lines.

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Differences in treatment solution affect the efficiency of keratin extraction in cultured human squamous cell carcinomas, malignant melanomas, and melanocytes. Using an aqueous solution that is excellent for cultured cells, we focused this study on the expression of keratin subunits in the spontaneously immortalized human keratinocyte cell line HaCaT. We extracted several keratin (K) subunits, namely K4, K7, K8, K15, K17, and K18, and ATP synthase alpha-chain, in addition to those previously reported by Boukamp et al. (J Cell Biol 1988;106:761-771) in human HaCaT keratinocytes. In particular, K8 and K18 subunits, which are related to tumorigenesis, may be very important subunits within the specificities of immortalized HaCaT cells. Vimentin, which is frequently co-expressed in cultured epithelial cell lines, was not expressed.

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Recently we demonstrated that the keratin 17 (K17) content exceeded the K16 content in most follicular tumors, in comparison with non-follicular epithelial skin tumours by two-dimensional gel electrophoresis (2-DE), densitometry and immunohistochemistry. At present the origin of basal cell carcinoma (BCC) is unknown. So, based on the above results, we studied keratin expression in eight cases of BCC, in order to analyze tumor differentiation by both biochemical and immunohistochemical methods. Biochemically, using 2-DE and immunoblotting, stratified epithelial keratins K5/K14 and large amounts of K17 were present in all cases. Simple epithelial keratins K8 and K19 were expressed in all and half of the cases, respectively. However, hyperproliferative associated keratins (K6/K16) and keratinized keratins (K1/K10) were detected in only a few cases. Immunohistochemical studies using frozen sections with chain-specific antikeratin monoclonal antibodies against K1, K7, K8, K10, K14, K16, K17, K18 and K19 showed that BCC tumor cells reacted positively with antibodies against K8, K14, K17 and K19, but did not react, or were rarely positive with K1, K7, K10, K16 and K18 antibodies. Predominant expression of K17 and the frequent expression of K8 and K19, with little K6/K16 and K1/K10 expression are the characteristic features of BCC, suggesting that BCC is differentiated towards undifferentiated follicular epithelia, most probably hair bulge cells.

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Eccrine syringofibroadenoma (ESFA) is a rare disorder which shows differentiation toward the eccrine sweat apparatus. There is a controversy concerning the pathogenesis and precise differentiation of this tumor. We report a case of ESFA and its differentiation pattern by an analysis of cytokeratin expression. Using paraffin-embedded materials, histopathological and immunohistochemical studies were performed. Staining patterns of the luminal, peripheral, and inner cells of the tumor strands closely matched or mimicked those of the luminal, outer and intermediate cells of the normal eccrine dermal duct, respectively. The case of ESFA reported revealed a pattern of differentiation suggestive of an eccrine duct origin.

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With the exception of two cases, keratin is not expressed in cultured human melanoma cells. Using 2D-PAGE, immunological and electron microscopic analyses, we found keratin subunits in five established cultured cell lines derived from primary, recurrent and metastasized melanomas. The keratin subunits were composed of K1, K5, K10, K14, K15 and K18 in all cell lines examined, together with vimentin. In addition, K8, K16 and K18 expression were demonstrated in recurrent and metastasized cell lines. The results of the present and our previous study [Katagata Y, et al. J Dermatol Sci 1996;13:219-227] indicate that expression of keratin in melanoma cells may be a universal phenomenon. A specific increase in the proportion of K5 among the keratin subunits was suggestive of the nature of melanoma cells. Moreover, we detected two polypeptides that migrated on 2D-PAGE at positions which did not correspond to those of any keratin subunit. The amino acid sequences of these two polypeptides were determined; one was the human ATP synthase alpha-chain but the other did not match any known polypeptide in our homology search.

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Keratin expression in cultured malignant melanoma cells has been studied only rarely. Moreover, no studies have reported of universality of keratin expression in human malignant melanoma cells. In this study, therefore, we analyzed keratin expression in eight cell lines. Using a low-salt aqueous solution without high salt and Triton X-100, as a washing buffer for keratin extraction, followed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and immunological analysis, we demonstrated keratin expression in all eight human malignant melanoma cell lines. The keratin polypeptide expressions common to all melanoma cells were K1, K5, K10 and K14. In addition, K8, K13, K17 and K18, respectively, were detected in individual cells. A measure of keratin expression universality in malignant melanoma cells may have implications regarding their invasive and metastatic behaviors, co-expressed with vimentin.

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We have reported that an upstream stimulatory factor (USF) binding site is functional in transcription of the heme oxygenase-1 gene. In this study, we examined the role of USF in the induced state. By transient expression analyses with the chloramphenicol acetyl-transferase gene, we found that the USF binding site plays an important role in the induction of rat heme oxygenase-1 by cadmium, but not by hemin. To elucidate the role of USF, we prepared USF-rich nuclear extracts from control and cadmium-treated rat liver. On electrophoretic mobility shift assay using control nuclear proteins, one slowly migrating band was detected, whereas using nuclear proteins of cadmium-treated rat liver, two fast migrating bands were detected. The molecular masses of the two subunits of USF prepared from cadmium-treated rat liver were approximately 34 kDa as determined by UV cross-linking and subsequent SDS-PAGE, while the two subunits of native USF were 43 kDa and 44 kDa. DNase I footprinting analysis revealed that both the nuclear proteins bound to the same region including the USF binding site. We therefore suppose that cadmium causes some structural changes in the two proteins of USF and that the altered USF participates in the effective initiation of transcription of the rat heme oxygenase-1 gene.

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Specimens of trichilemmal cyst, malignant trichilemmoma, keratoacanthoma, and epidermal cyst were examined to characterize keratin peptides in hair-follicle-derived tumors. Keratins were extracted from the specimens and analyzed by two-dimensional gel electrophoresis and densitometry; the results were then compared with those for normal epidermis, the outer root sheath of hair follicles, psoriatic epidermis, and various nonfollicular cutaneous epithelial tumors. The specific nonfollicular tumors examined were squamous cell carcinoma, Bowen disease, actinic keratosis, eccrine porocarcinoma, and sebaceous carcinoma. Immunohistochemistry also was performed with a few anti-keratin monoclonal antibodies. As a general rule, K6 and K16 were expressed in hyperproliferative conditions, such as epidermal tumors, and K17 was coexpressed in the same lesions. The ratio of K16 to K17 in many epithelial skin tumors has been unclear until now. K17 content exceeded K16 content in most follicular tumors, whereas in almost all the nonfollicular tumors and the psoriatic epidermis, K17 levels were less than or about equal to K16 levels. There was a significant difference in the ratio of K16 to K17 between follicular and nonfollicular skin tumors. These results indicate that alterations in the content of these keratins may be associated with follicular differentiation.

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Formalin-fixed and paraffin-embedded tissue specimens of six cases of extraocular sebaceous carcinoma were studied immunohistochemically with eight anti-keratin monoclonal antibodies, 34 beta B4, 35 beta H11, Ks13.1, Ks19.1, PKK1, LP34, KL1 and AE1. The staining patterns of sebaceous carcinoma were compared with those of normal sebaceous glands and other skin cancers which should be distinguished from sebaceous carcinoma histopathologically. The other skin cancers compared were eccrine porocarcinoma, malignant clear cell hidradenoma, extramammary Paget's disease with underlying adenocarcinoma, malignant trichilemmoma, and squamous cell carcinoma. Most cases of sebaceous carcinoma were stained with 35 beta H11, Ks19.1, LP34, KL1 and AE1, while normal sebaceous glands were positive only with 35 beta H11, LP34, KL1 and AE1. By immunostaining, sebaceous carcinoma was distinguishable from extramammary Paget's disease with underlying adenocarcinoma, squamous cell carcinoma, malignant trichilemmoma, and eccrine porocarcinoma, but was not clearly distinguishable from malignant clear cell hidradenoma. These findings demonstrate that sebaceous carcinoma shows positive reactions with antibodies to simple epithelial keratin, probably as a result of neoplastic transformation, and that immunohistochemical examination using anti-keratin monoclonal antibodies is useful in distinguishing sebaceous carcinoma from several other skin cancers.

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Recently K1 keratin peptide (K1, 68 kDa) was found to be present in two kinds of cultured human squamous cell carcinomas (HSCs) using a low-salt aqueous solution, rather than the high-salt solution containing Triton X-100 employed by many researchers up until now. To confirm whether this phenomenon is universal in cultured HSCs we analyzed K1 peptide in four other kinds of HSCs using the same procedures. Moreover, the K1 peptide detected was a little unusual with respect to solubility versus urea concentration. Epidermal K1 peptide is usually solubilized by 6-8 M urea and reductant; however, the K1 peptide in cultured HSCs was about 80-90% extracted by 1-2 M urea in a stepwise extraction procedure. This finding may have important implications regarding evaluation of keratin extracted from normal epidermal and cultured keratinocytes.

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Keratin specificity analyses of eight anti-keratin antibodies (34 beta B4 (K1), 35 beta H11 (K8), Ks 13.1 (K13), Ks 19.1 (K19), PKK1, LP34 (CK1), KL1 and AE1) using keratin protein derived from normal thigh epidermis, normal parotid gland and a human squamous cell carcinoma cell line (HSC-5) were performed, and compared with those described in the data sheets. The reactivities of LP34, KL1 and PKK1 were markedly different from those mentioned in the data sheets. The immunostaining pattern of these antibodies in normal skin using formalin-fixed and paraffin-embedded tissue specimens was also examined. The staining patterns of suprabasal keratinocytes (K1, K13, CK1 and KL1 positive), basal cells of the epidermis (PKK1 and AE1 positive), inner cells of the ducts (K8, K13, CK1, KL1 and AE1 positive) and secretory cells of sweat glands (K8, K19, PKK1, KL1 and AE1 positive), mature cells (K8 and KL1 positive) and peripheral cells (CK1, KL1 and AE1 positive) of sebaceous glands and outer root sheaths (PKK1, CK1, KL1 and AE1 positive) were specific. Thus, we conclude that the differentiation of epidermis and skin appendages is possible by immunostaining with these eight anti-keratin antibodies using formalin-fixed and paraffin-embedded tissue specimens with proper protease pretreatment.

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The largest keratin peptide (K1, 68KD) has not been detectable in cultured human squamous cell carcinomas. However, quite recently, the K1 peptide was clarified to be present in two kinds of cultured HSC by using a low salt aqueous solution, rather than the high salt and Triton X-100 employed by many previous researchers (Biochem. Biophys. Res. Commun., 182, 1440-1445, 1992). To determine whether this phenomenon is common or not in cultured HSCs, I further demonstrated the K1 peptide by extracting it with two different buffers and by 2D-PAGE, immunological techniques, and Northern blot analysis, using another kind of HSC. Until now, keratin extraction has been done using high salt/Triton X-100 solution, during which K1 peptide may be removed because it has developed an affinity with the buffer. Many investigators may have therefore overlooked it.

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