Signal transduction and mechanisms of cell death. Sixth colloquium on cellular signal transduction, 17 January 1997, Heidelberg, Germany.
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Animals↗
Biomedical subjects
P Boukamp
Publications and source records attributed to P Boukamp.
Telomerase activity in the regenerative basal layer of the epidermis inhuman skin and in immortal and carcinoma-derived skin keratinocytes.
Cellular senescence is defined by the limited proliferative capacity of normal cultured cells. Immortal cells overcome this regulation and proliferate indefinitively. One step in the immortalization process may be reactivation of telomerase activity, a ribonucleoprotein complex, which, by de novo synthesized telomeric TTAGGG repeats, can prevent shortening of the telomeres. Here we show that immortal human skin keratinocytes, irrespective of whether they were immortalized by simian virus 40, human papillomavirus 16, or spontaneously, as well as cell lines established from human skin squamous cell carcinomas exhibit telomerase activity. Unexpectedly, four of nine samples of intact human skin also were telomerase positive. By dissecting the skin we could show that the dermis and cultured dermal fibroblasts were telomerase negative. The epidermis and cultured skin keratinocytes, however, reproducibly exhibited enzyme activity. By separating different cell layers of the epidermis this telomerase activity could be assigned to the proliferative basal cells. Thus, in addition to hematopoietic cells, the epidermis, another example of a permanently regenerating human tissue, provides a further exception of the hypothesis that all normal human somatic tissues are telomerase deficient. Instead, these data suggest that in addition to contributing to the permanent proliferation capacity of immortal and tumor-derived keratinocytes, telomerase activity may also play a similar role in the lifetime regenerative capacity of normal epidermis in vivo.
Cell Line, Transformed↗
Step-wise progression in human skin carcinogenesis in vitro involves mutational inactivation of p53, rasH oncogene activation and additional chromosome loss.
Two mechanisms relevant for skin carcinogenesis in man are mutational inactivation of p53 and oncogenic activation of c-rasH gene. Previously, we transfected c-rasH oncogene into human skin keratinocytes (HaCaT) with u.v.-typic mutations in both p53 alleles, which produced benign and malignant tumorigenic clones, expressing similar amounts of mutant Ras protein. Here we show that neither the ras integration site nor the karyotypic changes affects the formation of the benign or malignant tumorigenic phenotype. From the original malignant HaCaT-ras clone we took single human chromosomes, carrying the c-rasH oncogene and transferred them by microcell mediated chromosome transfer into genetically different untransfected nontumorigenic HaCaT cells. This novel approach identified the genetic background of the recipient cell as a critical determinant for the resulting tumor phenotype. Exhibiting similar oncogene expression, microcell hybrids from early passage cells remained nontumorigenic or formed benign tumors, while those with more cytogenetic aberrations (later passages) and loss of > 1 copy of chromosome 15 became malignant. Since aberrations in chromosome 15 were also detected in three of five human skin carcinoma lines this study provides evidence that p53 and c-rasH mutations are early events of human skin carcinogenesis, while loss of gene(s) on chromosome 15 is a late event.
Cell Line↗
Transdifferentiation induced by gene transfer.
While for many tissues the differentiation process is well characterized, little is known about 'master switch' genes determining a specific differentiation pathway and having the potential to induce this process in a cell designed for a different differentiation pathway. Based on heterokaryon and 5-aza-cytidine-induced hypomethylation experiments, the muscle determination gene MyoD1 was identified and isolated, which was shown to induce myogenic differentiation even in cells of ectodermal lineage. Since transdifferentiation studies could also be performed in drosophila in vivo by 'false' expression of developmental genes, it is tempting to speculate that experimentally induced transdifferentiation mimics processes during embryonic development and tissue maturation.
Animals↗
Differentiation and tumor progression.
Clinical and experimental experience indicate that differentiation and malignancy are inversely correlated. However, more recent experimental studies using mouse and human keratinocyte systems have demonstrated that complete or even substantial loss in overall epithelial differentiation is not a prerequisite for malignant growth of cancer cells. Major defects in differentiation are also not a prerequisite for premalignant stages, in particular for cell immortalization, which is considered an early and essential step in the transformation process. Moreover, progressive dedifferentiation, often associated with advanced tumor stages, is also found in immortalized cell lines which are, however, nontumorigenic. On the other hand, malignant cell lines may have maintained a high degree of their normal differentiation program and sensitivity to differentiation modulators. However, to date no transformed keratinocyte cell lines with completely normal differentiation have been observed. Since epidermal keratinization is a very complex process involving many different parameters and is fully expressed only under in vivo conditions, an exact and quantitative comparison of such ill-defined phenomena (differentiation and malignancy) is still problematic. Obviously, both phenomena are under separate control and not causally linked. Nevertheless, a better understanding of factors and mechanisms regulating differentiation and of their disturbance in carcinogenesis would offer new possibilities to design novel tumor therapeutic strategies in the field of differentiation therapy.
Biomarkers↗
International conference on cell interactions in malignancy, development and differentiation. Heidelberg, Germany, April 1995.
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Animals↗
Association of deficient DNA repair during G2 phase with progression from benign to malignant state in a line of human skin keratinocytes transfected with ras oncogene.
Human skin keratinocytes after malignant neoplastic transformation by infection with Kirsten murine sarcoma virus (KiMSV) or transfection with pSV2 ras (containing an activated c-Ha-ras oncogene) showed a DNA repair deficiency(ies). The repair deficiency was manifest as an abnormally high frequency of chromatid breaks and gaps persisting after X-ray-induced DNA damage inflicted during the G2 phase of the cell cycle. Non-tumorigenic control cells at that time were clearly repair-efficient. By analyzing benign and malignant tumorigenic HaCaT-ras clones, we could exclude ras p21 oncoprotein expression as the causal mechanism for repair deficiency, since both clone types expressed similar levels of the mutated protein and only the malignant tumorigenic cells showed repair deficiency. The results suggest that mutated p21 ras provided the human keratinocytes with a growth advantage in vivo (benign tumor growth), but acquisition of repair deficiency is required for progression from benign to malignant state.
Animals↗
Expression and function of connexin in normal and transformed human keratinocytes in culture.
We have studied the gap junctional intercellular communication (GJIC) of immortalized and tumourigenic human keratinocyte cell lines and of a spontaneously immortalized non-tumourigenic and a highly differentiating keratinocyte cell line (HaCaT) as the control. In homologous cultures, the GJIC capacity of five squamous cell carcinoma-derived cell lines was 1-27% that of the HaCaT cells. Ha-ras-transfected HaCaT cells with tumourigenic potential and an SV40 DNA-immortalized cell line had markedly reduced GJIC capacities. Northern analysis and immunohistochemistry showed that connexin (Cx) 43 is the major gap junction protein expressed in the communicating cells. They do not express Cx 26 or 32. The low or absent communication observed in certain cell lines was due in some to a lack of Cx 43 gene expression, but in others to aberrant localization of the gap junction protein. GJIC of these cell lines, as well as that of primary normal human epidermal keratinocytes, was susceptible to 12-O-tetradecanoylphorbol-13-acetate-mediated inhibition. Moreover, GJIC of HaCaT cells and their tumourigenic derivatives is Ca(2+)-dependent. These results, when compared with those previously obtained for mouse keratinocyte cell lines, reveal that GJIC of human keratinocytes was correlated to the degree of differentiation and is controlled in a similar way to that of murine keratinocytes. Aberrant GJIC seems to be a common feature of human and murine skin carcinogenesis.
Carcinoma, Squamous Cell↗
Binding and activation of plasminogen at the surface of human keratinocytes.
Plasmin is thought to be involved in the pericellular proteolysis of the human epidermis under physiological and pathological conditions. Plasmin is provided by activation of the proenzyme plasminogen. We have explored in vitro whether plasminogen is bound and activated at the keratinocyte surface, a possible mechanism for providing plasmin in the pericellular space. Plasminogen and plasmin could be eluted from the surface of keratinocytes grown in serum-containing medium. When plasminogen was added to cultured keratinocytes it was activated by cell-associated urokinase-type plasminogen activator. The activation required plasminogen binding to the cell surface. Plasminogen binding by keratinocytes was saturable and proceeded in a time- and concentration-dependent manner. Surface-bound plasmin was rapidly displaced from the surface into the culture supernatant. When compared to plasmin in solution surface-bound plasmin was relatively protected from interaction with the specific inhibitor alpha 2-antiplasmin. Addition of exogenous plasmin or plasmin generation by the keratinocyte-associated plasminogen activators was ensued by the detachment of adherent keratinocytes in culture. Along the same line, plasmin counteracted keratinocyte adhesion to fibrin-coated but not to collagen-coated culture plates. The findings indicate that plasmin may be generated in the pericellular space of keratinocytes and may interfere with the adhesion to particular extracellular substrates.
Cell Adhesion↗
In vitro transformation and tumor progression.
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Animals↗
"Trans-differentiation" from epidermal to mesenchymal/myogenic phenotype is associated with a drastic change in cell-cell and cell-matrix adhesion molecules.
Cells of the human keratinocyte line HaCaT were shifted to a mesenchymal/myogenic phenotype (DTHMZ cells) by MyoD1 transfection, 5-aza-2' deoxycytidine treatment, and selection for reduced adhesion on plastic. Since this correlated with loss of stratification (inability to form a multilayered tissue), we determined the status of cell-cell and cell-matrix adhesion molecules involved in epidermal morphogenesis. Expression of desmosomal proteins (plakoglobin, desmoglein, desmoplakin) and uvomorulin was no longer detectable at the mRNA and protein level in the DTHMZ cells while both HaCaT cells and malignant variants (transfected with c-Ha-ras oncogene) expressed uvomorulin in vitro and in transplants in vivo, the latter even in invasively growing tumor nodules. Furthermore, HaCaT cells stained positive for the integrin subunits beta 1, alpha 2, alpha 3, and alpha 5, typical for cultured keratinocytes. In contrast, the putative fibronectin receptor alpha 5 beta 1, common also in fibroblasts, was the only integrin showing strong staining in DTHMZ cells. The integrin subunits alpha v and a6, clearly expressed at the mRNA level, weakly stained HaCaT cultures and led to a dotlike fluorescence in DTHMZ cells, possibly representing focal adhesion plaques. The respective integrin status correlated well with the growth behavior on different matrices. While HaCaT cells readily attached and proliferated on collagen (type I), fibronectin-coated, and laminin-coated collagen gels, DTHMZ cells formed monolayers only on fibronectin-coated collagen. This was, however, not sufficient to allow stratification in vivo. Altogether, the status of adhesion molecules in DTHMZ cells more likely reflects that seen in mesenchymal cells as compared to the pattern of keratinocytes displayed by HaCaT cells. Moreover, since the DTHMZ cells were clearly HaCaT descendants, the results support our hypothesis of a "trans-differentiation" process from an epidermal (HaCaT) to a mesenchymal/myogenic phenotype (DTHMZ).
Cadherins↗
p53 mutations in human immortalized epithelial cell lines.
Although rodent cells have been immortalized following transfection with a mutant p53 gene, the role of p53 in the immortalization of human cells is unknown. Therefore, human epithelial cell lines were examined for p53 mutations in exons 4-9 which include the evolutionarily conserved regions. A spontaneously immortalized skin keratinocyte cell line, HaCat, and three ras-transfected clones, have a p53 mutational spectrum that is typical of ultraviolet light induced mutations. A normal finite lifespan cell strain (184) and two benzo[a]pyrene immortalized mammary epithelial cell lines derived from 184 (184A1 and 184B5) contain wild type p53 sequences in exons 4-9, although elevated levels of nuclear p53 indicate an alteration in the stability of the normally transient protein. Wild type p53 was found in human bronchial, esophageal and hepatic epithelial cells immortalized by SV40 T antigen gene and human renal epithelial cells immortalized by adenovirus 5. BEAS-2B, an SV40 T antigen immortalized bronchial epithelial cell line and two subclones, have a germline polymorphism at codon 47. Inactivation of p53 by mechanisms such as mutation or complexing with proteins of DNA tumor viruses appears to be important in the immortalization of human epithelial cells.
Base Sequence↗
Progressive stages of "transdifferentiation" from epidermal to mesenchymal phenotype induced by MyoD1 transfection, 5-aza-2'-deoxycytidine treatment, and selection for reduced cell attachment in the human keratinocyte line HaCaT.
The ability of the myogenic determination gene (MyoD1) to convert differentiating human keratinocytes (HaCaT cell-line) to the myogenic pathway and the effect of MyoD1 on the epidermal phenotype was studied in culture and in surface transplants on nude mice. MyoD1 transfection induced the synthesis of myosin, desmin, and vimentin without substantially altering the epidermal differentiation properties (morphology, keratin profile) in vitro nor epidermal morphogenesis (formation of a complex stratified squamous epithelium) in surface transplants, demonstrating the stability of the keratinocyte phenotype. 5-Aza-CdR treatment of these MyoD1-transfected cells had little effect on the cultured cells but a morphologically unstructured epithelium was formed with no indications of typical cell layers including cornification. Since prevention of epidermal strata in transplants was not accompanied by blocked epidermal differentiation markers (keratins K1 and K10, involucrin, and filaggrin), the dissociation of morphogenesis and expression of these markers argues for independently controlled processes. A subpopulation of less adhesive cells, isolated from the 5-aza-CdR treated MyoD1-transfectants, had lost most epithelial characteristics in culture (epidermal keratins, desmosomal proteins, and surface-glycoprotein Gp90) and had shifted to a mesenchymal/myogenic phenotype (fibroblastic morphology, transactivation of Myf3 and myogenin, expression of myosin, desmin, vimentin, and Gp130). Moreover, the cells had lost the ability to stratify and remained as a monolayer of flat elongated cells in transplants. These subsequent changes from a fully differentiated keratinocyte to a mesenchymal/myogenic phenotype strongly argue for a complex "transdifferentiation" process which occurred in the original monoclonal human epidermal HaCaT cells.
Animals↗
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↗
The radiosensitivity of human keratinocytes: influence of activated c-H-ras oncogene expression and tumorigenicity.
We have investigated the gamma-ray sensitivity of several activated c-H-ras (EJ) containing clones that have been established after transfection of the spontaneously immortalized non-tumorigenic human keratinocyte cell line HaCaT. The clones were grouped according to their tumorigenic potential after subcutaneous injection into nude mice, and fell into three classes: Class I clones A-4 and I-6 are non-tumorigenic and express very low levels of c-H-ras mRNA and no mutated ras protein (p21); Class II clones I-5 and I-7 grow to large (benign) epidermal cysts, express intermediate to high c-H-ras mRNA and variable levels of mutated ras p21 protein with clone I-5 expressing little and clone I-7 expressing high levels of p21; Class III clones II-3 and II-4 grow to solid squamous cell carcinomas, express high c-H-ras mRNA and high level of mutated p21 ras protein similar to clone I-7. Comparison of the single-hit multitarget or linear-quadratic survival curve parameters, and survival at 2 Gy (S2) indicate that there appears to be no general correlation with either activated c-H-ras expression level or tumorigenic potential, and increased radioresistance.
Animals↗
The early genes E6 and E7 of cancer associated human papilloma viruses as targets of tumor suppression?
We have transplanted HPV 18 positive nontumorigenic hybrid cells and tumorigenic hybrid segregants as well as nontumorigenic and tumorigenic human keratinocytes immortalized by HPV 16-DNA transfection (HPK cells and HPK-ras cells) into nude mice and grown them under in vivo conditions for different periods of time. By analyzing gene expression at the mRNA level of the early viral genes E6/E7 and a number of cytoskeletal cellular genes we wanted to test the hypothesis that the nontumorigenic phenotype of these cells in vivo may be determined by the specific downregulation of expression of the oncogenic E6/E7 genes by the product(s) of tumor suppressor gene(s). The results obtained showed that: 1) The nontumorigenic hybrid cells (in contrast to tumorigenic segregants) stopped to proliferate about 3 days after transplantation. At this time E6/E7 gene expression was already drastically reduced, whereas at day 2 expression was still high. This suppression specifically affected the HPV 18 E6/E7 genes and preceded cell death by at least 10 days. 2) The same specific suppression of HPV E6/E7 gene expression occurred during in vivo growth of the nontumorigenic HPK cells. These cells gave rise to transiently growing cysts, but had retained the in vivo differentiation potential of normal keratinocytes (orderly expression of cytokeratins 1 and 10, involucrin and filaggrin). HPV 16 E6/E7 gene expression was very low and clearly restricted to a small subset of basal cells, thus showing an inverse relationship to terminal differentiation. In contrast, in the tumors induced by HPK-ras cells E6/E7 gene expression extended from the basal cells into suprabasal cells and terminal differentiation was retarded. These results support the hypothesis that during in vivo growth tumor suppressor gene(s) negatively regulated HPV E6/E7 gene expression resulting in the stop of proliferation of the nontumorigenic cells.
Animals↗
Activation of the plasminogen activator system in a keratinocyte cell line (HaCaT) by alkyl sulfates.
A human keratinocyte cell line (HaCaT) was analysed for plasminogen activator activity. By immuno-capture and immuno-inhibition studies we obtained evidence that HaCaT cells synthesize and secrete urokinase-type plasminogen activator (uPA) and tissue-type PA (tPA). Zymographic analysis by using fibrin agar indicator gels indicated that part of the PA activity in culture supernatants of the HaCaT line is complexed with putative PA inhibitors (PAI). Alkyl sulfates of different chain length were found to have a strong stimulatory effect on PA activity in HaCaT culture supernatants. Our findings are discussed in view of the known skin-irritating effects of alkyl sulfates in vivo.
Cell Line↗
Expression of epithelial antigens Exo-1 and EPM-1 in human epidermal keratinocyte maturation and benign and malignant neoplasia.
Exo-1, a polar neutral glycolipid, and EPM-1, a high molecular weight glycoprotein, are developmental antigens of human epithelial cells, initially described as components both on the cell surface and in secretions of gastrointestinal epithelial and respective tumors. In order to assess the biological significance of both antigens for epithelial cell differentiation and neoplastic transformation, their expression during human skin development and benign and malignant neoplasia was analyzed in fresh frozen tissue specimens of skin biopsies and of human epidermal keratinocytes growing in experimental model systems. Antigen expression was assessed immunohistochemically with specific monoclonal antibodies. During fetal development Exo-1 was temporarily expressed in intermediate cells but was absent in normal adult human skin. Exo-1 expression reemerged in neoplasias, both benign and malignant, but was restricted to spinous-like differentiated cells. Similarly, Exo-1 was not expressed in transplants of normal keratinocytes mimicking the normal epidermis but was clearly visible in differentiated areas of transplants of malignantly transformed keratinocytes. EPM-1 appeared first in basal epidermal cells in the second half of gestation and remained detectable in the stratum basale of adult skin. While squamous cell carcinomas continued to express EPM-1, it was not detectable in basal cell epitheliomas and in normal epidermis after invasion by neuroectodermal tumor cells. In experimental models, EPM-1 was present in the basal layers of normal human keratinocytes and of transformed keratinocytes with benign growth characteristics whenever a well stratified and keratinized epidermis-like epithelium had formed in transplants. In transformed keratinocytes with malignant growth behavior, EPM-1 was expressed irregularly, as in squamous cell carcinomas in situ. Thus, expression of Exo-1 is a marker for an early embryonic differentiation pathway of human keratinocytes and in adult tissue reveals abnormal differentiation associated with certain stages of hyperproliferation. EPM-1 expression is part of developmental programs and is influenced by microenvironmental interactions and alterations of tissue homeostasis.
Antigens↗