Robert H. Rice

Keratinocyte-specific transglutaminase of cultured human epidermal cells: Relation to cross-linked envelope formation and terminal differentiation

The predominant form of the cross-linking enzyme, transglutaminase, in cultured normal human epidermal keratinocytes, is found in cell particulate material and can be solubilized by nonionic detergent. It elutes as a single peak upon either anion-exchange or gel-filtration chromatography. Monoclonal antibodies raised to the particulate enzyme cross-react with one of two transglutaminases in the cell cytosol. The second cytosolic transglutaminase, which has distinct kinetic and physical properties from the first, does not cross-react and is not essential for formation of the keratinocyte cross-linked envelope in vitro. The anti-transglutaminase antibodies stain the more differentiated layers of epidermis in a pattern similar to that given by anti-involucrin antiserum. These observations support the hypothesis that the transglutaminase so identified is involved in cross-linked envelope formation in vivo.

Interactions of heme proteins with hydrogen peroxide: Protein crosslinking and covalent binding of benzo[a]pyrene and 17β-estradiol

This work reveals two biochemical effects of hydrogen peroxide treatment on hemoglobin, myoglobin, and cytochrome c. First, these heme proteins rapidly formed covalently crosslinked dimers and polymers detectable by detergent gel electrophoresis. Second, when treated in the presence of radioactive benzo[a]pyrene or 17 beta-estradiol, the heme proteins became covalently labeled. Nonheme proteins exhibited both cross-linking and radioactive labeling upon peroxide treatment in the presence but not the absence of heme protein or free hemin. Benzoyl peroxide or glucose and glucose oxidase effectively replaced direct addition of hydrogen peroxide. These results indicate that adventitious peroxidase activity expressed by oxygen carrying and electron transport proteins yields active oxygen species that can damage these heme proteins and nearby macromolecules, a possible biochemical mechanism for the lethal and other deleterious intracellular effects of peroxide.

Immunoperoxidase staining for involucrin. A potential diagnostic aid in cervicovaginal pathology

Involucrin, a protein subunit of keratinocyte cross-linked envelopes, is a distinctive marker for suprabasal differentiation in stratified squamous epithelium. Immunoperoxidase staining for involucrin was used to evaluate paraffin sections of tissue obtained by colposcopically directed biopsies of infectious, metaplastic, and dysplastic lesions of the cervix and vagina. Areas of normal squamous epithelium, papillary and flat condyloma acuminatum, and mature and immature squamous metaplasia showed positive staining in 99 per cent of samples lacking significant inflammation and in 60 per cent of those with moderate or severe inflammation. In contrast, only 19 per cent of the squamous cell dysplasias, even those without much inflammation, showed positive staining, and no area with moderate or severe inflammation showed positive staining. These findings indicate that expression of involucrin is modulated by cellular pathologic features and microenvironment. We suggest that immunoperoxidase staining for involucrin may be useful in distinguishing mild dysplasia from immature metaplasia and flat condyloma in some biopsy specimens in which routine histologic examination yields an indeterminate diagnosis.

Papillomavirus infection of the cervix. III: Relationship of the presence of viral structural proteins to the expression of involucrin

Forty-two cervical biopsies with cervical intraepithelial neoplasia were compared with respect to the expression of human papillomavirus (HPV) structural proteins and the expression of the cellular structural protein involucrin, a marker of suprabasal squamous differentiation. HPV structural protein and involucrin expression displayed an inverse correlation with the severity of dysplasia. Both of these proteins were detected in 11 of 28 cases (39%) of mild and moderate dysplasia, but in only two of 14 (14%) cases of severe dysplasia. This difference was statistically significant (p less than 0.001). The presence of HPV was also associated with expression of involucrin in the full thickness of the epithelium, including the basal layer, and an altered staining pattern in the more superficial cells, particularly the koilocytotic cells. These findings support the hypothesis that squamous differentiation is required for the expression of viral structural proteins and that HPV infection begins in the basal epithelium. The study also demonstrates the utility of involucrin staining in differentiating virus-induced cytologic atypia from true neoplasia.

Involucrin: A Constituent of Cross-Linked Envelopes and Marker of Squamous Maturation

The coordinated program of terminal differentiation in epidermis yields a superficial layer of tough dead squames well suited to the protective function of the integument. These mature cells consist primarily of insoluble disulfide-bonded keratin tonofilaments, but also exhibit a “cornified envelope” immediately beneath the plasma membrane. This structure, consisting of protein, is resistant to keratinolytic agents (alkali, detergent and reducing agent) and organic solvents, but is sensitive to proteolytic digestion (Matoltsy and Balsamo 1955; Sun and Green 1976). The chemical stability of the envelope is attributable to a high degree of e-(γ-glutamyl)lysine cross-linking arising from cellular transglutaminase activity (Sugawara 1977; Rice and Green 1977). Envelopes are conspicuous in hair and nail samples boiled in the presence of sodium dodecyl sulfate (SDS) and reducing agent (Rice and Green, unpublished), where their rigid interlocking convolutions contribute to the exceptional cohesiveness of the cells in these appendages (Green et al. 1982). Envelopes are present on the surface of all the stratified squamous epithelia of the human and have been observed in the cornified layer of epidermis of mammals, birds, reptiles and amphibians but not fish (Matoltsy 1977).

Melittin-stimulated arachidonic acid metabolism by cultured malignant human epidermal keratinocytes

Upon melittin stimulation, cultured SCC-13 keratinocytes release prostaglandins E2, F2 alpha, 6-keto-F1 alpha, thromboxane B2, leukotriene B4, and 6-sulfido-peptide-containing leukotrienes (SRS) into serum free medium. Release of prostaglandins E2, F2 alpha, and SRS, normalized to cell protein, is 3- to 10-fold higher from rapidly growing than confluent cultures. Cells growing with hydrocortisone in the medium produce approximately twice the level of the cyclooxygenase-mediated metabolites PGE2 and PGF2 alpha as those without hydrocortisone, but similar levels of the lipoxygenase-mediated metabolite SRS. The results demonstrate the potential utility of squamous carcinoma lines for investigating biochemical pathways of arachidonic acid metabolism in keratinocytes.

CHARACTERIZATION OF THE CALCIUM SENSITIVITY OF DIFFERENTIATION IN SCC-13 HUMAN SQUAMOUS CARCINOMA CELLS

SummaryThe sensitivity to calcium of the human squamous carcinoma cell line, SCC-13, was demonstrated and characterized. Cultures grown to confluence in the presence of 0.2 to 2 mM calcium had approximately 10-fold higher levels of particulate transglutaminase activity and envelope competence than those grown in low calcium (0.025 to 0.05 mM) medium. Raising the calcium from 0.025 to 1.8 mM induced expression of this enzyme and of competence over the course of a week. Conversely, for cultures grown to confluence in 1.8 mM calcium, subsequent reduction of calcium to 0.025 mM resulted in a substantial decline in transglutaminase over a similar time period. Immunoprecipitable transglutaminase was clearly identifiable in cultures grown in 1.8 mM calcium-containing medium but not in those grown in low calcium medium or in the presence of retinoic acid, suggestive of regulation at the level of mRNA accumulation or translation rather than posttranslational modification.

Keratinocyte transglutaminase: regulation and release.

Recent improvements in the serial culture of keratinocytes (Rheinwald, 1980; Allen-Hoffman and Rheinwald, 1984) now permit detailed analysis of the differentiation program in this cell type (Green, 1979). To study the program at a molecular level requires identification and characterization of specific marker proteins, regulation of whose synthesis and utilization are important in the cell function. In addition to the high content of keratin (and related cytoskeletal elements), the intracellular transglutaminase is responsible in part for the distinctive structural cohesiveness of mature squames. Thus, a critical feature of terminal differentiation is the formation of an envelope of cross-linked protein immediately beneath the plasma membrane, a transglutaminase-mediated process carried out by keratinocytes in culture (Sun and Green, 1976; Green, 1977; Rice and Green, 1977 and 1978). The cross-linking is elicited by elevation of calcium ion available to the enzyme and results in utilization of involucrin (Rice and Green, 1979; Simon and Green, 1985) and several other protein substrates (Simon and Green, 1984).

Evaluation of squamous epithelium in adenoacanthoma and adenosquamous carcinoma of the endometrium: immunoperoxidase analysis of involucrin and keratin localization.

A study was undertaken to determine whether immunoperoxidase stains for keratin and involucrin, the latter a protein present in cells of stratified squamous epithelium that have differentiated beyond the basal stage, distinguish any differences in squamous cells present in the adenoacanthoma from those in the adenosquamous carcinoma of the uterine corpus. Forty-eight tumors were studied, of which 33 were adenoacanthomas and 15 adenosquamous carcinomas. The patients with adenoacanthomas were slightly younger (mean 61.5 vs. 64.5 years) and had tumors that were generally better differentiated than the adenosquamous carcinomas. The squamous epithelium in every tumor, regardless of histologic type, stained positively for keratin. There were no obvious differences in staining when tumors were stratified for histologic type, grade, or location within the tumor. The glandular portion of both tumor types stained irregularly, but nonetheless positively, for keratin in 71% of the cases. Involucrin was detected in 57% of adenoacanthomas and 87% of adenosquamous carcinomas. The deeper or more central portion of the squamous morules stained only if the more superficial or peripheral areas were positive. The extent of the involucrin staining was less in the adenosquamous carcinomas than in the adenoacanthomas. The glandular component of the tumors did not stain for involucrin. It is concluded that no qualitative differences in the staining reactions with respect to keratin and involucrin distinguish the adenoacanthomas from the adenoaquamous carcinoma. These findings support the argument that the adenoacanthoma and adenosquamous carcinoma represent a spectrum of squamous differentiation in a single tumor type.

Cross-Linked Envelopes: Keratinocyte Transglutaminase

Membrane-bound transglutaminase plays a key role in the formation of cross-linked envelopes during keratinocyte terminal differentiation. The enzyme can be solubilized from the particulate fraction of cultured epidermal cells by nonionic detergent Alternately, it can be released in a soluble form by treatment of the particulates with hydroxylamine, indicating that esterified fatty acid serves as a membrane anchor. Facilitating study of structure-function relations, the released enzyme can be purified nearly to homogeneity by affinity chromatography on a column of B.C1 monoclonal antibody covalentfy linked to Sepharose 4B. In SCC-9 neoplastic human keratinocytes, expression of the transglutaminase markedly increases as the cultures arrive at confluence, approximately a week before their involucrin content becomes appreciable. Arrest of cell growth with α-difluoromethylomithine prior to confluence leads to the increased expression of transglutaminase, but involucrin synthesis is largely prevented. Thus, the cells appear to enter a state of intermediate differentiation at confluence which is stabilized by arrest of DNA synthesis, thereby uncoupling the differentiation program. However, when the cells are grown in medium supplemented with low serum concentrations (0.1–0.4%), reduced cell growth and saturation densities are accompanied by reduced rates of increase in transglutaminase activity. Of potential assistance in assays of transglutaminase in crude extracts, especially those of low specific activity, reductively methylated casein is shown to give six-fold higher incorporation of labelled putrescine than unmodified casein.