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D Breitkreutz

Publications and source records attributed to D Breitkreutz.

At least 37 records · Page 2Linked to original sources
PubMed1993

Phenotypic modulation of human hair matrix cells (trichocytes) by environmental influence in vitro and in vivo.

Trichocytes, i.e. precursor cells of the hair cortex and medulla, isolated from plucked human scalp hair follicles (HF) were propagated on feeder layers of post-mitotic human dermal fibroblasts (HDF). Cell isolates from five HF routinely yielded about 0.5-1 x 10(5) cells within 3 weeks. When grown as 'surface epithelia' in vitro (on dermal equivalents exposed to air), trichocytes organized into stratified epithelia largely reminiscent of epidermis with regard to both tissue architecture and localization of epidermal differentiation products (keratins K1 and K10, involucrin, filaggrin). However, when HDF in the collagen matrix were replaced by dermal papilla cells (DPC) epidermoid differentiation was largely prevented while still allowing growth and stratification. Epidermal differentiation (keratinization) was virtually complete when trichocytes grown on collagen gels with HDF were transplanted onto nude mice; this was apparent by tissue organization, expression of K1 and K10 and the nearly regular epidermal localization of involucrin. In addition, the deposition of basement membrane components (laminin, type IV collagen, bullous pemphigoid antigen) at the epithelium-collagen interface further increased and was more regular in transplants than in vitro. Cells embedded in Matrigel together with HDF developed large spheroidal structures with inward-directed differentiation and all the epidermal markers found in 'surface' cultures, while only small keratinizing spheroids formed without HDF. In this system co-culture of trichocytes with DPC suppressed almost completely epidermal keratinization. Although typical hair proteins were not detectable, our data clearly demonstrate that: (1) bona fide trichocytes inherit the options for alternative directions of differentiation, and (2) external (in part mesenchymal cell-mediated) influences play a pivotal role in this determination.

Animals↗
PubMed1991

Experimental modulation of the differentiated phenotype of keratinocytes from epidermis and hair follicle outer root sheath and matrix cells.

Follicles of human anagen hair were separated into morphologically distinct compartments (by sequential trypsinization and microdissection) for the biochemical and immunological analysis of keratins as differentiation markers to diagnose the type of epithelial differentiation. While outer root sheath contained throughout the "soft" (cyto)keratins K5, 6, 14, 16, and 17, and hair cortex contained exclusively a set of acidic and basic "hard" alpha-keratins (consistent up to the hair tip), in inner root sheath and hair cuticle peptides related or derived from suprabasal epidermal keratins K1 and 10 were detected. These keratin profiles served as in vivo correlates for the evaluation of type and degree of differentiation achieved by the respective isolated epithelial cells, comparing different growth or culture conditions. Cultures of ORS cells and hair matrix cells (PHS cells) as well as normal keratinocytes were initiated using postmitotic human dermal fibroblasts as efficient feeder cells. On lifted collagen gels populated with HDF ("surface" cultures), ORS and PHS cells formed stratified epithelial expressing epidermal differentiation markers such as keratins K1 and 10, involucrin, and filaggrin. Compared with NEK "surface" cultures, balance between growth and differentiation was better maintained by both follicular cell types. In contrast, epidermal tissue homeostasis was largely normalized in transplants on nude mice regardless of the epithelial cell type, apparent from orderly tissue structure, regular distribution of keratin K10, filaggrin, and involucrin, and distinct continuous deposition of basement membrane components at the epithelium-collagen interface. Embedded in Matrigel (on top of HDF collagen gels) ORS cells and NEK formed spheroids exhibiting inward-directed epidermoid differentiation, increasing with time. All epidermal maturation products found in "surface" cultures were likewise expressed, and again differentiation greatly outbalanced proliferation in spheroids of NEK but not of ORS cells. PHS cells embedded together with HDF in Matrigel produced similar spheroids as ORS cells. Size of spheroids and degree of epidermoid differentiation were dramatically reduced when HDF were replaced by follicular DP cells, demonstrating the crucial role of the mesenchymal "companion" cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell Differentiation↗
PubMed1991

Epidermal morphogenesis and keratin expression in c-Ha-ras-transfected tumorigenic clones of the human HaCaT cell line.

Several tumorigenic (benign and malignant) clones have been raised from the human epidermal cell line HaCaT after transfection with the c-Ha-ras oncogene (val 12) (P. Boukamp et al., Cancer Res., 50: 2840-2847, 1990). In culture, these HaCaT-ras clones expressed epidermal differentiation markers, such as keratins K1 and 10, at high density or upon depletion of retinoic acid. Accordingly, as HaCaT cells, the clones formed well-differentiated stratified epithelia synthesizing K1 and 10 in surface transplants, while simple and internal epithelial keratins seen in culture were suppressed (as upon retinoic acid depletion in vitro). In transplants of HaCaT cells, in contrast to those of normal keratinocytes, K1 appeared prematurely already in basal cells, while K10 localized rather normally in the suprabasal position. Keratins 1 and 10 were also synthesized in transplants of HaCaT-ras clones (again K1 preceding K10), but both generally shifted toward upper layers. This was particularly evident in thicker transplants of malignant clones. Staining for both keratins persisted "suprabasally" in invasive tissue masses, and this corresponded to their marked expression in solid carcinomas (after s.c. injection), seen by immunofluorescence and two-dimensional gel electrophoresis. Thus, notwithstanding some variations, differentiation potential was not significantly reduced in these clones disregarding levels of ras oncogene expression and malignant properties.

Cell Line↗
PubMed1991

Restoration of the epidermal phenotype by follicular outer root sheath cells in recombinant culture with dermal fibroblasts.

In order to better understand how outer root sheath (ORS) cells are able to reepithelialize superficial skin wounds, the level of epidermal differentiation achieved by isolated ORS cells in vitro was determined. Using postmitotic human dermal fibroblasts (HDF) as efficient feeder cells, large numbers of ORS cells from individual follicles were generated. Passaged ORS cells were grown exposed to air on HDF-populated collagen gels in the CRD device (Noser and Limat, In vitro 23, 541-545, 1987) which allows histiotypic tissue organization. In such recombinant organotypic cultures, ORS cells developed distinct epidermal strata comparable to interfollicular keratinocytes (NEK). Ultrastructurally, desmosomes and intermediate filaments increased in number toward the epithelial surface and small keratohyalin (KH) granules (but no large irregular KH granules as in NEK) were abundant, adjacent to an electrondense stratum corneum. Also, synthesis of epidermal suprabasal keratins (K1 and 10;2D gels) was lower in ORS cultures, but clearly visible suprabasally by immunofluorescence along with other epidermal markers (involucrin, filaggrin, surface glycoprotein gp80, pemphigus vulgaris antigen). Basement membrane components (laminin, type IV collagen, bullous pemphigoid antigen) were detectable in both ORS and NEK in these assays. Thus, phenotypic expression was largely comparable, but, whereas terminal differentiation (keratinization) was progressing in NEK cultures limiting their lifespan, this seemed to be better controlled in ORS cultures and viable cell layers persisted resulting in longer survival time.

Adult↗
PubMed1991

Transcription of the human loricrin gene in vitro is induced by calcium and cell density and suppressed by retinoic acid.

We have previously shown that loricrin is a major component of the cornified cell envelope (CE) expressed late in epidermal differentiation in the granular layers of normal skin. Normal human keratinocytes were cultured under various conditions and loricrin mRNA levels were assessed at various time points. Only Ca++ concentrations above 0.1 mM Ca++ were permissive for the expression of lori-crin mRNA. Maximal mRNA levels were found at 0.35 mM Ca++ and a critical cell density appeared to be required for optimal accumulation of loricrin transcripts. Retinoic acid (RA) at 10(-7) to 10(-9) M completely blocked Ca+(+)-induced loricrin mRNA synthesis when applied simultaneously. Furthermore, addition of RA to cultures already exposed to higher Ca++ levels resulted in the complete loss of loricrin mRNA within 48-72 h. So far, no other components of the CE have been shown to be suppressed by RA. However, similar patterns of expression were reported for filaggrin, a matrix protein also expressed late in epidermal differentiation. Therefore, we compared the mRNA levels of loricrin and filaggrin and found them to change in parallel in response to the various culture conditions. These results suggest that Ca++, cell density, and RA are crucial regulators of loricrin expression in vitro and that the transcriptional control of loricrin and filaggrin expression in the epidermis are closely coordinated.

Blotting, Northern↗
PubMed1990

Mesenchyme-mediated and endogenous regulation of growth and differentiation of human skin keratinocytes derived from different body sites.

In culture, keratinocytes generally express aberrant growth and differentiation programs, which are largely normalized in cell transplants. In order to study the underlying regulatory phenomena and to distinguish between intrinsic properties and external factors, different in vitro and in vivo models have been applied using human keratinocytes from foreskin and trunk skin. When transplanted onto nude mice, keratinocytes reformed a regular epithelium with expression of the differentiation markers, keratins K1 and K10, involucrin and filaggrin. Tissue homeostasis improved in later transplants, as made apparent by coexpression and regular distribution of K1 and K10. Since this was achieved in transplants, whether in contact with mesenchyme or separated by collagen matrix, renormalization was obviously mediated by diffusible factors. In vitro, the host-mesenchymal influence could largely be mimicked by recombining organotypic cultures (keratinocytes on lifted collagen gels) with de-epidermized dermis, but tissue homeostasis was apparently not achieved. Comparing keratinocytes from trunk skin and foreskin, differences observed in situ persisted in isolated cells and reconstituted tissues. The hyperproliferative character of foreskin epidermis, with its less-pronounced stratum granulosum, was maintained in recombinant cultures and transplants along with the expression of keratin K13 (typical for foreskin in situ) irrespective of the type of mesenchyme. Thus, we could demonstrate with these model systems that: (a) the regulation of keratinocyte growth and differentiation is mesenchyme-dependent; (b) it is mediated by diffusible factors; but that (c) differences between epidermis of different body sites are also controlled by intrinsic programs.

Adolescent↗
PubMed1990

Keratins 1 and 10 or homologues as regular constituents of inner root sheath and cuticle cells in the human hair follicle.

Morphological maturation of the inner root sheath (IRS) and cuticle of the human hair follicle reveals analogies to differentiation processes in other keratinizing epithelia. Detailed biochemical analysis of respective differentiation products, however, has been largely restricted by their low solubility. Herein we provide further evidence for the existence of K1 and K10-derivatives in IRS and hair cuticle based on protein analysis of isolated fractions and immunofluorescence in situ, substantiating our earlier data (Stark, H. J., et al. Differentiation 35, 236-248 (1987)). Extracts from both compartments showed on two-dimensional (2D)-polyacrylamide gels a group of presumptive K1 and K10-turnover products in a wide pI (basic to acidic) and Mr range (56,000-65,000), named IC-I to III and IC-IV, respectively. These components (also found in nail plate) reacted with specific antibodies (to K1 and K10) on Western blots. Weak but distinctive radiolabeling of presumptive precursor spots close to authentic K1 and K10, respectively, and their presence in lower follicle fractions (distant from infundibulum) largely precluded epidermal contamination. Two-dimensional tryptic peptide maps of excised 2D spots from the IC-I to III series revealed high homology to K1, and those from IC-IV components to K10. Immunodetection in frozen sections was improved by trypsin pretreatment and showed distinguished staining for K1 and K10 in IRS ranging from the lower bulbus region up to the "keratinizing zone" of the follicle. Above, the reaction was abruptly abolished which coincides with ultrastructural "melting" of distinct filaments in the intracellular matrix. Thus, our data suggest that differentiation in these follicular compartments (IRS and cuticle) might follow common principles of keratinization.

Fluorescent Antibody Technique↗
PubMed1989

Density-dependent modulation of synthesis of keratins 1 and 10 in the human keratinocyte line HACAT and in ras-transfected tumorigenic clones.

The spontaneous human keratinocyte line HaCaT and c-Ha-ras oncogene-transfected cell clones are capable of expressing an unusually broad spectrum of keratins, not observed so far in epithelial cells. This expression is, however, strongly modulated by environmental conditions, including cell density. Both cells of the nontumorigenic HaCaT line and the tumorigenic HaCaT-ras clones, I-7 and II-3 (giving rise to benign and malignant tumors, respectively), constitutively expressed the keratins K5, K6, K14, K16 and K17, which are also common in cultures of normal keratinocytes. In addition keratins K7, K8, K18 and K19, generally associated with simple epithelia, were synthesized (to a most pronounced extent in sparse cultures), while keratins K4, K13 and K15 appeared at confluence, presumably with the onset of stratification. Moreover, in both HaCaT and HaCaT-ras clones the epidermal "suprabasal" keratins, K1 and K10, were expressed in conventional submerged cultures (at normal vitamin A levels), markedly rising with cell density, but not strictly correlated with the degree of stratification. This property was maintained in HaCaT cells up to the highest passages. According to immunofluorescence, this was due to increasing numbers of strongly stained cells, and not due to a gradual increase in all cells. Most strikingly, there was a significant delay in the appearance of K10 compared to K1, and this dissociation of expression was most evident in dispase-detached cell sheets (submerged cultures) and organotypic cultures of the ras clones (grown at the air-liquid interface). While on frozen sections bright staining for K1 was seen in some basal and virtually all suprabasal cell layers, K10 was largely restricted to the uppermost layers. Thus, obviously synthesis of K1 and K10 can be regulated independently, although generally in this given sequence. The apparent compatibility of K1 synthesis with proliferation and particularly the extended delay of K10 expression (as a postmitotic event) might be causally related to altered growth control and as such imply the significance of this disturbance. Finally, the highly preserved epidermal characteristics, in terms of expression of keratins (and other differentiation markers [5]) and their regulation, makes these cell lines excellent candidates for studying external modulators of differentiation and also underlying molecular mechanisms.

Antibody Specificity↗
PubMed1988

Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.

In contrast to mouse epidermal cells, human skin keratinocytes are rather resistant to transformation in vitro. Immortalization has been achieved by SV40 but has resulted in cell lines with altered differentiation. We have established a spontaneously transformed human epithelial cell line from adult skin, which maintains full epidermal differentiation capacity. This HaCaT cell line is obviously immortal (greater than 140 passages), has a transformed phenotype in vitro (clonogenic on plastic and in agar) but remains nontumorigenic. Despite the altered and unlimited growth potential, HaCaT cells, similar to normal keratinocytes, reform an orderly structured and differentiated epidermal tissue when transplanted onto nude mice. Differentiation-specific keratins (Nos. 1 and 10) and other markers (involucrin and filaggrin) are expressed and regularly located. Thus, HaCaT is the first permanent epithelial cell line from adult human skin that exhibits normal differentiation and provides a promising tool for studying regulation of keratinization in human cells. On karyotyping this line is aneuploid (initially hypodiploid) with unique stable marker chromosomes indicating monoclonal origin. The identity of the HaCaT line with the tissue of origin was proven by DNA fingerprinting using hypervariable minisatellite probes. This is the first demonstration that the DNA fingerprint pattern is unaffected by long-term cultivation, transformation, and multiple chromosomal alterations, thereby offering a unique possibility for unequivocal identification of human cell lines. The characteristics of the HaCaT cell line clearly document that spontaneous transformation of human adult keratinocytes can occur in vitro and is associated with sequential chromosomal alterations, though not obligatorily linked to major defects in differentiation.

Aneuploidy↗
PubMed1987

Keratins of the human hair follicle: "hyperproliferative" keratins consistently expressed in outer root sheath cells in vivo and in vitro.

Keratins produced by morphologically distinct compartments of the human hair folicle (hHF) were analysed and compared to those produced by cultured hHF and interfollicular keratinocytes. Five of the major keratins, the basic keratins nos. 5 and 6 (apparent mol. mass 60 and 58 kDa) and the acidic keratins nos. 14, 16, and 17 (51, 49 and 48 kDa), could be labelled in intact hHF and were found in all fractions of the outer root sheath (ORS). The other major keratins, which were not labelled under these conditions (basic-neutral hHbI and -II; 60-62 kDa and acidic hHaI and -II; 40-42 kDa) were associated with hair shaft (hHS) both in the follicle and, virtually unchanged, in the distal part of the hair. Another, previously undescribed, group of proteins with keratin-like properties exhibiting a broad pI-spectrum (basic to slightly acidic: hIC-I, -II, -III, 64-67 kDa; distinctly acidic: hIC-IV, about 54 kDa) was detected in isolated inner root sheath (IRS), in the cuticular material shed from denuded hHS, and also in nail plates. In our experiments only ORS cells grew readily in culture irrespective of their origin from peripheral (mesenchyme-adjacent) or more central ORS-cell layers. In contrast to keratinocytes from interfollicular epidermis (IFE) the cultured ORS cells expressed a keratin set virtually identical to that expressed in vivo. This set also closely resembled that expressed by IFE keratinocyte cultures. The identity of the respective keratins (nos. 5, 6, 14, 16, and 17) present in all these cells in vivo and in vitro was confirmed by tryptic peptide mapping. The data indicated that the microenvironment (in situ) directs the differentiation of ORS cells in a manner comparable to the way it is directed by conventional culture conditions, with consistent expression of the "basal" and "hyperproliferative" set of keratins. This, however, does not exclude the possibility that other types of environmentally induced response may occur, as seen for example during the reepithelialization of superficial skin wounds by ORS cells.

Cells, Cultured↗
PubMed1986

Environmental induction of differentiation-specific keratins in malignant mouse keratinocyte lines.

Four spontaneously transformed keratinocyte lines (HELP I-IV) were raised from primary cultures of mouse epidermal cells grown on gas-permeable (Petriperm) dishes. Although tumorigenic, these cell lines still expressed the differentiated phenotype under mesenchymal influence in vivo in a fashion similar to normal cells and in contrast to previous observations on other transformed cell lines. Initially, after transplantation onto adult mice, HELP cells generally formed well organized ortho-keratinizing epithelia closely resembling those of normal epidermal cells. Later, dysplastic epithelia and papilloma-like structures developed and cells invaded subcutaneous host tissue. When injected subcutaneously into newborn syngeneic mice, all four cell lines gave rise to differentiated carcinomas at high frequency. Keratinized metastases were detected in the lung with HELP I, albeit at low frequency. Although the four HELP cell lines differed morphologically and biochemically in their degree of ortho-keratinization, no inverse relationship to their malignant potential was evident. In contrast to cell cultures, HELP transplants and tumors expressed epidermis-type "suprabasal" keratins. Metabolic labeling and electrophoresis on one and two-dimensional gels revealed both the basic 67 kilodaltons (kDa) and acidic 58 kDa components, including presumptive derivatives analogous to those observed in epidermal stratum corneum. However, associated with alterations in tissue architecture, the spatial control of keratin expression was gradually lost in papilloma-like and invading transplants and tumors, as demonstrated by indirect immunofluorescence microscopy (IIF). Thus, while cell differentiation appeared virtually normal, the progressive disturbances in tissue differentiation indicate important changes in the responsiveness of these malignant keratinocytes to environmental conditions.

Animals↗
PubMed1984

Proteolytic modification of acidic and basic keratins during terminal differentiation of mouse and human epidermis.

Keratins from the living cell layers of human and neonatal mouse epidermis (prekeratins) have been compared to those from the stratum corneum (SC keratins). Human and mouse epidermis contained four prekeratins, two of each keratin subfamily: type II basic (pI 6.5-8.5; human 68 kDa, 60.5 kDa and mouse 67 kDa, 60 kDa) and type I acidic (pI 4.7-5.7; human 57 kDa, 51 kDa and mouse 58 kDa, 53 kDa,). While all four were present in equal amounts in adult human epidermis, two (67 kDa basic, 58 kDa acidic) were more prominent in neonatal mouse epidermis. Preliminary results with cell fractions (basal, spinous and granular) indicated that quantitative differences were a function of morphology, basal cells containing the smaller member of each subfamily and granular cells the larger. Mouse stratum corneum extracts contained four keratins (three in human): type II neutral-acidic (pI 5.7-6.7; human 65 kDa and mouse 64 kDa, 62 kDa) and type I acidic (pI 4.9-5.4; human 57.5 kDa, 55 kDa and mouse 58.5 kDa, 57.5 kDa). In both species, one-dimensional and two-dimensional peptide mapping (with V8 protease and trypsin respectively) indicated that while all four prekeratins were distinct gene products, similarities existed in the type II basic and the type I acidic keratin subfamilies. A strong homology also existed between type II SC keratins and the larger basic (type II) prekeratin (human 68 kDa and mouse 67 kDa) and between type I SC keratins and the larger acidic (type I) prekeratin (human 57 kDa and mouse 58 kDa). These results indicate a precursor-product relationship within each keratin subfamily, between SC keratins and the prekeratins abundant in the adjacent granular layer. This differentiation-related keratin processing was similar in mouse and human epidermis, and may represent a widespread phenomenon amongst keratinising epithelia.

Animals↗
PubMed1984

Differentiation specific functions in cultured and transplanted mouse keratinocytes: environmental influences on ultrastructure and keratin expression.

Keratinocytes from neonatal mouse back skin, growing in primary culture (PEC) under conventional conditions (immersed), exerted a reduced programme of differentiation as indicated by cell morphology and organisation, ultrastructure and keratin composition. Four major keratins were found in cytoskeletal extracts (mol.wt. 60K, 59K, 53K, 49K) of primary cultures, together with a minor 51K protein and some residual actin. This "culture-type" keratin profile remained stable and little variation was observed after repeated treatment with various agents, such as 12-0-tetradecanoylphorbol-13-acetate (TPA), retinoic acid (RA) or dimethylsulphoxide (DMSO). The profile was unaltered by long-term growth of primary cultures in low Ca2+ (0.1 mM) medium or on 3T3 feeder-layers. Nevertheless, TPA, RA and low Ca2+ did alter the morphology and filament architecture (as observed by indirect immunofluorescence microscopy with anti-keratin antibodies). Comparison of keratinocytes from primary culture with basal cells isolated from epidermis revealed similarities in electrophoretic profile, with the 60K and 53K keratins being common to both. The other in vivo keratins had a characteristic spatial distribution; 67K and 58K keratins were present in suprabasal cells (spinous and granular), while 64K, 62K, 58.5K and 57.5K keratins were present only in stratum corneum. None of these keratins were found in cultured cells grown under regular conditions. Several morphological features of epidermal differentiation could be restored by the growth of PEC on collagen gels exposed to the atmosphere ("organotypic" culture) without influencing the keratin profile. Almost complete restoration of epidermal function was achieved after transplantation of PEC onto adult syngeneic mice. In this in vivo environment, well-structured epithelia developed which resembled interfollicular epidermis. Restoration of both typical ultrastructure and in vivo type keratin expression occurred within 2 or 3 weeks. Thus, although keratinocytes in primary culture differ considerably from those in vivo, they have not irreversibly lost the capacity for complete differentiation.

Animals↗
PubMed1983

Completion of the amino acid sequence of the alpha 1 chain from type I calf skin collagen. Amino acid sequence of alpha 1(I)B8.

The complete amino acid sequence of the 279-residue CNBr peptide CB8 from the alpha 1 chain of type I calf skin collagen is presented. It was determined by sequencing overlapping fragments of CB8 produced by Staphylococcus aureus V8 proteinase, trypsin, Endoproteinase Arg-C and hydroxylamine. Tryptic cleavages were also made specific for lysine by blocking arginine residues with cyclohexane-1,2-dione. This completes the amino acid sequence analysis of the 1054-residues-long alpha (I) chain of calf skin collagen.

Amino Acid Sequence↗
PubMed1983

Growth and differentiation characteristics of transformed keratinocytes from mouse and human skin in vitro and in vivo.

The altered phenotypic expression and chromosomal characteristics of mouse and human malignant keratinocyte lines have been studied in vitro and in vivo (in comparison with normal primary cultures). The cell lines exhibited different morphologic aspects that are probably more related to their respective degree of differentiation than to different stages in malignancy. Although all cell lines studied were deficient in some aspects of keratinization, certain basic structural and biochemical features were maintained, and these may serve as valid criteria for the identification of their epithelial nature. The altered expression of keratin proteins and morphologic differentiation can be modulated under in vivo growth conditions, but they cannot be reverted toward normality. Chromosomal alterations (in number and structure) occur early and are highly indicative criteria for malignancy, even though no tumor-specific aberrations have been identified. Two new approaches for evaluating characteristics of abnormal growth and differentiation in vitro, and of invasiveness in vivo, have been developed and have proved sensitive test methods for identifying malignant cells. While several abnormalities in growth and differentiation of cell lines in vitro are highly indicative of their malignant nature, the final proof that they are tumor cells still requires in vivo assay. The transplantation assay for studying cellular invasiveness not only improves the sensitivity of in vivo malignancy tests but has also proved to be a valuable model system for elucidating the modulation of differentiation by external influences.

Animals↗
PubMed1983

Preservation of morphological, functional, and karyotypic traits during long-term culture and in vivo passage of two human skin squamous cell carcinomas.

Two cell lines (SCL-I and SCL-II) derived from squamous cell carcinomas of human skin were investigated during 4 years in culture. Both lines were tumorigenic in nude mice, and cells could be recultivated from xenografts. Growth in agar remained poor, but both cell lines developed abnormal stratified epithelial structures in organotypical cultures. The morphological and particularly ultrastructural characteristics remained typical in both cultures and xenografts. Keratinization slightly decreased, but nude mouse tumors differentiated as the original tumors, and this was reflected in keratin expression. Six major polypeptides (Mr 61,000, 57,000, 54,000, 51,000, 49,000, and 45,000) were similarly identified in both tumors and cell lines, also after animal passage, which was further substantiated by two-dimensional gel electrophoresis, but quantitative variations were found with different growth conditions. A distinct keratin cytoskeletal network was visualized in both lines by immunofluorescence, but only a few cells in SCL-II also expressed vimentin. Flow cytometry demonstrated 2c DNA stem lines for original tumors and derived lines. Early passages were hypodiploid by cytogenetic analysis of banded chromosomes. In SCL-I, a shift to tetraploidy occurred before passage 20 and remained stable throughout. In SCL-II, an incomplete shift to near tetraploidy and a stem line deviation were apparent, but later passages, nude mouse tumors, and cells recultured therefrom were hypodiploid (2c) again. Chromosome studies further revealed distinct stable marker chromosomes which showed additional structural aberrations with time in culture and after animal passage. Thus, phenotypically and genotypically, each squamous cell carcinoma and its derived cell line were distinct, and characteristics were preserved over long time periods in vitro and through in vivo passage.

Animals↗