Biomedical subjects

P Boukamp

Publications and source records attributed to P Boukamp.

At least 55 records · Page 3Linked to original sources

PubMed1990

c-Ha-ras oncogene expression in immortalized human keratinocytes (HaCaT) alters growth potential in vivo but lacks correlation with malignancy.

Spontaneously immortalized human skin keratinocytes (HaCaT) were transfected with the c-Ha-ras (EJ) oncogene via a plasmid construct which also contained the selectable neomycin gene. Clones were selected on the basis of G418 resistance. Those clones that had stable integrants of Ha-ras fell into 3 classes with respect to tumorigenicity. Class I clones were nontumorigenic, i.e., formed nodules which rapidly regressed. This phenotype is identical to that seen with parental HaCaT cells. Class II clones formed slowly growing, highly differentiated cystic or papillomatous-type benign tumors, and class III clones formed highly differentiated, locally invasive squamous cell carcinomas. The clones of all three classes exhibited similar morphology and growth potential in culture and retained the ability to reconstitute an epidermis-like stratified epithelium in transplantation experiments. Only the malignant clones showed locally invasive growth. Both the benign and the malignant clones exhibited higher levels of ras integration and variable levels of mutated p21 protein product. Thus, expression of the cellular Ha-ras oncogene in these human epithelial cells significantly altered growth regulation, resulting in varying degrees of growth potential in vivo, ranging from benign to malignant tumors. However, no direct correlation was seen between high levels of p21 expression and malignant growth.

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

Suppression in vivo of human papillomavirus type 18 E6-E7 gene expression in nontumorigenic HeLa X fibroblast hybrid cells.

The E6 and E7 genes of the cancer-associated human papillomavirus (HPV) types 16 (HPV16) and 18 (HPV18) can induce cell immortalization in vitro in normal human keratinocytes. This, however, is not associated with tumorigenicity in vivo. On the other hand, tumorigenicity of HPV18-positive HeLa cervical carcinoma cells can be suppressed by fusion of HeLa cells with normal human keratinocytes or fibroblasts. We have addressed the question of whether suppression of tumorigenicity in HeLa x fibroblast hybrid cells might be due to a reduced ability of these cells to express the HPV18 E6-E7 genes in vivo. Nontumorigenic hybrid cells and tumorigenic hybrid segregants were transplanted as organotypical cultures or injected subcutaneously into immunocompromised mice and were analyzed for HPV18 E6-E7 gene expression by RNA-RNA in situ hybridization. The tumorigenic hybrid cells showed a continuous and invasive growth that was associated with high levels of HPV18 E6-E7 mRNAs at all time points examined. In contrast, the nontumorigenic hybrid cells stopped cell proliferation approximately 3 days after transplantation. At this time they expressed the E6-E7 genes at low levels, whereas at day 2 high expression levels were observed. However, the mRNA levels of the cytoskeletal genes beta-actin and vimentin remained high for at least 14 days, demonstrating that inhibition of growth and of HPV18 E6-E7 gene expression was not due to cell death. These results suggest that growth inhibition of the nontumorigenic HeLa x fibroblast hybrid cells in vivo might be caused by suppression of HPV18 E6-E7 gene expression and are compatible with the idea of an intracellular surveillance mechanism for HPV gene expression existing in nontumorigenic cells.

Blotting, Northern↗

PubMed1989

Sensitization of human keratinocytes to killing by parvovirus H-1 takes place during their malignant transformation but does not require them to be tumorigenic.

To investigate the antineoplastic activity of parvoviruses, proliferating normal human epidermal cells and a series of established keratinocyte cell lines derived from squamous cell carcinomas or transformed in vitro, were compared for the outcome of H-1 virus infection. All established keratinocyte cell lines were more sensitive to killing by H-1 virus than normal epidermal cells, although to varying extents. Using a step-wise procedure for malignant transformation in vitro, we found that sensitization of transformed epidermal cells to H-1 virus can be dissociated from the acquisition of a tumorigenic phenotype. Thus, spontaneously- or SV40-immortalized human keratinocytes were moderately and highly sensitive to H-1 virus, respectively, and could be made tumorigenic by Harvey-ras oncogene transfection without a major change in their susceptibility to the virus. The capacity of human keratinocytes for replicating and expressing H-1 virus DNA appears to be a revealer of cellular alterations that take place in at least some pathways to malignant transformation but that may be insufficient to confer a tumorigenic potential.

Cell Survival↗

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

Allele-specific methylation of the human c-Ha-ras-1 gene.

The methylation status of individual c-Ha-ras-1 alleles in human cells was measured by Hpall/Mspl analysis of a polymorphic (VTR) region. The gene was extensively methylated in leukocytes and sperm, with the former exhibiting a highly specific methylation pattern. Individual ras alleles were differentially methylated at the VTR region in fetal fibroblasts and immortal cell lines. A new polymorphism in the 5'-flanking region of the gene was also detected. The presence or absence of an Xhol site showed a striking and complete concordance with the length of the VTR region and suggested that the site had been lost by a previous allele-specific mutation.

Alleles↗

PubMed1987

Effect of growth environment on spatial expression of involucrin by human epidermal keratinocytes.

Involucrin, the major protein precursor of the cornified envelope, is expressed during terminal differentiation of human keratinocytes, both in vivo and in vitro. In epidermis, the onset of synthesis is several layers above the basal layer, but in stratified cultures of keratinocytes on tissue culture plastic involucrin synthesis begins in the first suprabasal layer. To investigate the reason for this premature expression, the distribution of involucrin was studied in epidermis from different body sites, in organotypic cultures and in transplants of keratinocytes onto nude mice. We found that premature expression was not associated with poor morphological differentiation, because involucrin synthesis began immediately above the basal layer even when distinct basal, spinous, granular and cornified layers were formed in organotypic cultures recombined with dermis. The site of involucrin expression in culture did not depend on the number of cornified layers present. The only conditions which resulted in an upward shift in the site of synthesis were in 3-week old transplants on nude mice. We conclude that the site of onset of involucrin synthesis is not determined by the degree of morphological differentiation of the tissue, and discuss other factors which may be involved.

Adult↗

PubMed1987

Molecular and cytogenetic analysis of immortalized human primary keratinocytes obtained after transfection with human papillomavirus type 16 DNA.

A proliferating population of human foreskin keratinocytes (presently in the sixtieth passage) has been obtained after transfection with human papillomavirus (HPV) type 16 DNA. In contrast, the control cultures did not survive beyond the sixth passage. Cytogenetic analysis of cells taken from the twelfth passage revealed a heteroploid male karyotype. In approximately 50% of the cells a common marker chromosome was found, suggesting a clonal origin for at least part of the population. This is further substantiated by Southern blot analysis of cellular DNA which revealed oligomeric HPV 16 genomes integrated at a single site within the host DNA. RNA transcribed from the early region of the HPV 16 genome was identified in the cytoplasm. The immortalizing effect of HPV 16 DNA on human keratinocytes could be reproduced in a second experiment. Such cell lines represent an unique system to study the interaction of HPV with its natural target cell in vitro.

Cell Division↗

PubMed1985

Environmental modulation of the expression of differentiation and malignancy in six human squamous cell carcinoma cell lines.

Cell lines from six human squamous cell carcinomas exhibiting different degrees of differentiation and malignancy were studied under in vitro and in vivo growth conditions. The stability of phenotypic traits of these carcinoma cells and their sensitivity to environmental influences were analyzed to further elucidate the interdependency of differentiation and malignancy expressed under experimental conditions. In conventional (submerged) cultures the cell lines exhibited unique growth patterns with an individual but generally poor expression of differentiation (stratification). In a new organotypical culture assay where the cells grew on lifted collagen gels at the air-medium interface, three-dimensional structures were formed exhibiting organizational features and degrees of differentiation similar to those of the respective tumors. Both in tumors formed after s.c. injection of cells and in transplants (performed with silicone chambers on the dorsal muscle fascia) in nude mice, an enhancement of the individually distinct pattern of differentiation was observed. While anchorage independent growth was an unreliable marker for malignancy, all six lines were tumorigenic after s.c. injection into nude mice. However, the tumor yield (20 to 100%) and latency period (2 to 12 weeks) varied considerably. In contrast all lines exhibited (within 1 to 2 weeks) invasive growth in 100% of animals after transplantation onto the dorsal muscle fascia. All tumors (squamous carcinomas) and invading cells were identified as epithelial and as human by specific antibodies. The two new test systems, the organotypical culture assay in vitro and the transplantation assay in vivo, proved to be reliable and sensitive models also for human squamous carcinoma cells to analyze their differentiative and malignant potential. In comparing the individually maintained degrees of differentiation and malignancy in the different test systems, it was apparent that, opposite to the prevailing opinion, cell lines with the highest differentiation potential were at least as malignant as were the least differentiated ones.

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

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↗

PubMed1982

Phenotypic and genotypic characteristics of a cell line from a squamous cell carcinoma of human skin.

A human cell line (SCL-1) from a poorly differentiating cutaneous squamous cell carcinoma (SCC) ws studied through 20 passages during 2 years. Cells maintained their original morphology with low degree of keratinization, as indicated by light and electron microscopy. The keratin peptide pattern resembled the type in SCC tumors, and the corresponding filaments were detected by immunofluorescence at all passage levels. Cells did not grow in soft agar but formed tumor-like nodules in an "organotypic" culture assay (on lifted collagen gels) and grew invasively after transplantation to immunosuppressed inbred C3H mice. After injection into BALB/c nu/nu mice, tumors of SCC morphology were formed. The hypodiploid tumor stem-line was maintained for about 10 passages, when a shift to hyperploidy started, as determined by chromosome and DNA flow microfluorometric analyses. Two stable marker chromosomes (in 100 and 70% of the metaphases, respectively), involving chromosomes 7 and 9, strongly indicated a monoclonal origin of this cell line.

Aged↗

PubMed1981

Correlation of prekaratin peptides and ultrastructure in epithelial cells of human skin tumors in vivo and in vitro.

Prekeratin was reduced in human skin malignancies comparing Bowen's carcinoma (BC), basal cell carcinomas (BCC) and squamous cell carcinomas (SCC) with normal epidermis. This observation correlated with ultrastructural appearance and frequency of tonofilaments. Gel electrophoresis of tumor extracts revealed the decrease or loss of larger prekeratin peptides (65 to 68 K daltons) prominent in the epidermis. Biopsies, particularly from BC, resembled normal keratinocytes in culture with respect to their prekeratin patterns (48 to 61 K daltons). The pattern was most different, and prekeratin lowest, in SCC and derived cultures. In BCC-cultures, however, prekeratin (almost identical to keratinocytes) and filament formation significantly exceeded the respective tumor levels.

Bowen's Disease↗

PubMed1979

Simultaneous expression of two different types of intermediate sized filaments in mouse keratinocytes proliferating in vitro.

The intermediate-sized filaments present in epidermal keratinocytes derived from mouse skin and in an established cell line (HEL) derived from spontaneous transformation of murine keratinocytes grown in vitro, have been examined by immunofluorescence microscopy, using antibodies directed against subunit proteins of different classes of intermediate-sized filaments, as well as by electron microscopy and gel electrophoresis of cytoskeletal preparations highly enriched in intermediate-sized filaments. The keratinocytes derived from neonatal skin, which are capable of only limited replication in vitro, show only a single type of intermediate-sized filaments, i.e., the tonofibril-like arrays of filaments containing prekeratin. HEL cells, which proliferate indefinitely in vitro, retain the tonofilament-like structures typical of differentiated epidermal cells but in addition display intermediate-sized filaments of the vimentin type, i.e., the filament system typically found in mesenchymal and mesenchyme-derived cells. We discuss the possibility that (i) the advent of vimentin-type filaments in epidermal cells in culture is related either to the transformed state or the in vitro growth conditions as such and (ii) other differentiated epithelial cells proliferating in vitro may have more than one system of intermediate-sized filaments.

Animals↗

PubMed1978

Characteristics of chemically transformed mouse epidermal cells in vitro and in vivo.

Primary cultures of epidermal cells from newborn mouse skin have been established. These cultures proliferate and express typical epidermal functions for limited time in vitro. The cultured cells keratinize and exhibit strong reactivity with an epidermis specific membrane antiserum. These cells can be transformed with DMBA either in standard cultures or more easily in cultures using 3T3 feeder cells. Transformed cells have increased proliferation rate. They do not pile up or form multilayered cultures like normal counterparts. They also keratinize less. When transformed cells were grown in the air on collagen gels they formed irregular clumps with central (horn-pearl) keratinization whereas normal controls formed stratified structures. Transofmred cells exhibited reduced reactivity with tissue specific membrane antiserum. There was masking or rearrangement of tissue specific membrane antigens with simultaneous exposition of new fetal-like antigens. Their reactivity with histocompatibility antisera was only slightly reduced. Transformed cells had both numerical and structural chromosome aberrations. They grew in soft agar and they induced tumors upon transplantation in the convenient host. When transplanted as cultures (on collagen) or as suspensions into the host, transformed cells were able to form epithelial cell cords invading the underlying mesenchyme with histological aspect typical of carcinoma. Cell cultures derived from in vivo DMBA induced tumors behaved like in vitro transformed cells.

9,10-Dimethyl-1,2-benzanthracene↗

PubMedDate not supplied

Telomerase is active in normal gastrointestinal mucosa and not up-regulated in precancerous lesions.

Telomerase is a ribonucleoprotein complex which, by de novo synthesized telomeric TTAGGG repeats, prevents telomere erosion. While telomerase is active in most cancers, conflicting results exist for normal tissues and premalignant lesions. To establish the telomerase status of normal gastrointestinal mucosa and to elucidate whether telomerase up-regulation is an early or late event in carcinogenesis, we determined the telomerase activity of 88 biopsies of normal mucosa from esophagus, stomach, and intestine and compared it with that of 21 samples of premalignant lesions and 6 adenocarcinomas using the telomere-repeat amplification protocol assay. Telomerase was found in all normal tissues, revealing most activity in esophagus (11 samples), followed by intestine (45 samples), and stomach (32 samples). In 53% of the stomach samples, enzyme activity could only be demonstrated when telomerase inhibitors were eliminated by a modified telomerase assay. In the 21 precancerous lesions (5 Barrett's esophagus, 3 stomach intestinal metaplasias, and 13 colorectal adenomas of type I/II dysplasia) a similar or even reduced telomerase activity was seen, while the adenocarcinomas showed high activity. These data demonstrate that telomerase activity is expressed in all epithelia along the gastrointestinal tract, thus confirming our previous hypothesis that telomerase is constitutively expressed in permanently renewing epithelia. Furthermore, activity was not increased in preneoplastic lesions, suggesting that telomerase up-regulation is a late event during carcinogenesis of the esophagus, stomach, and intestine.

Adult↗

PubMedDate not supplied

Matrix metalloproteinases-2,-3,-7,-9 and-10, but not MMP-11, are differentially expressed in normal, benign tumorigenic and malignant human keratinocyte cell lines.

In order to investigate the correlations between constitutive proteinase expression and the degree of tumorigenicity of cancer cells we have studied a model system of three keratinocyte cell lines. RT-PCR studies showed that the cell lines express the genes of matrix metalloproteinase-2, -3, -7, -9, -10 and -11, indicating that they are able to synthesize the corresponding enzymes. Actual MMP synthesis was proven by zymography and Western blotting. In conditioned media gelatinolytic activities or immunoreactive forms of MMP-2, -3, -7, -9, -10 and -11 were detected. The signal intensities showed that MMP secretion increases in the order HaCaT < A5 < or = II-4RT, whereas only MMP-11 is secreted by all cell lines in equal amounts. Intracellularly, enhanced levels of one or both of the tumorigenic variants were only found for MMP-3, -9 and -10, suggesting special functions of these intracellular MMP pools for the tumorigenic cell lines. For MMP-11 exclusive expression in stromal fibroblasts of tumor tissues is widely accepted; however, our results and three other recent reports demonstrate that this concept is not generally valid. In conclusion, the three keratinocyte cell lines investigated here represent an excellent model for studying constitutive expression and secretion of MMPs in correlation to the degree of in vivo tumorigenicity.

Base Sequence↗