Postmitotic Fibroblasts Research Articles

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Proliferative and morphologic characterization of buccal mucosal fibroblasts in areca nut chewers: A cell culture study

To isolate, culture and characterize fibroblasts from the buccal mucosa of areca nut chewers with and without oral submucous fibrosis (OSF). Primary fibroblast cultures were established by the collagenase disaggregation technique and the phenotypic and growth characteristics were studied. Cells cultured from OSF showed a statistically significant increase in both the post-mitotic fibroblast subpopulation and the population doubling time when compared with controls. There was a significant increase in the pro-fibrotic, post-mitotic subpopulation of fibroblasts in areca nut chewers with OSF.

The Experimental Measurement of the Relative Biological Effectiveness of Carbon Ions with Different Qualities

The relative biological effectiveness (RBE) of carbon ions with linear energy transfer (LET) of 172 keV/μm and 13.7 keV/μm were determined in this study. The clonogenic survival and premature terminal differentiation were measured on normal human fibroblasts AG01522C and NHDF after exposure of the cells to 250 kV X-rays and carbon ions with different qualities. RBE was determined for these two biological end points. The results showed that the measured RBE10 with a survival fraction of 10% was 3.2 for LET 172 keV/μm, and 1.33 for LET 13.7 keV/μm carbon ions. RBE for a doubling of post-mitotic fibroblasts (PMF) in the population was 2.8 for LET 172 keV/μm, and 1 for LET 13.7 keV/μm carbon ions. For the carbon ion therapy, a high RBE value on the Bragg peak results in a high biological dose on the tumour. The tumour cells can be killed effectively. At the same time, the dose on healthy tissue would be reduced accordingly. This will lighten the late effect such as fibrosis on normal tissue.

The Influence of Fractionation on Cell Survival and Premature Differentiation after Carbon Ion Irradiation

To investigate the influence of fractionation on cell survival and radiation induced premature differentiation as markers for early and late effects after X-rays and carbon irradiation. Normal human fibroblasts NHDF, AG1522B and WI-38 were irradiated with 250 kV X-rays, or 266 MeV/u, 195 MeV/u and 11 MeV/u carbon ions. Cytotoxicity was measured by a clonogenic survival assay or by determination of the differentiation pattern. Experiments with high-energy carbon ions show that fractionation induced repair effects are similar to photon irradiation. The RBE(10) values for clonogenic survival are 1.3 and 1.6 for irradiation in one or two fractions for NHDF cells and around 1.2 for AG1522B cells regardless of the fractionation scheme. The RBE for a doubling of post mitotic fibroblasts (PMF) in the population is 1 for both single and two fractionated irradiation of NHDF cells. Using 11 MeV/u carbon ions, no repair effect can be seen in WI-38 cells. The RBE(10) for clonogenic survival is 3.2 for single irradiation and 4.9 for two fractionated irradiations. The RBE for a doubling of PMF is 3.1 and 5.0 for single and two fractionated irradiations, respectively. For both cell lines the effects of high-energy carbon ions representing the irradiation of the skin and the normal tissue in the entrance channel are similar to the effects of X-rays. The fractionation effects are maintained. For the lower energy, which is representative for the irradiation of the tumor region, RBE is enhanced for clonogenic survival as well as for premature terminal differentiation. Fractionation effects are not detectable. Consequently, the therapeutic ratio is significantly enhanced by fractionated irradiation with carbon ions.

Co-Regulation of p16INK4a and Migratory Genes in Culture Conditions that Lead to Premature Senescence in Human Keratinocytes

Cellular stasis, also known as telomere-independent senescence, prevents many epithelial cells from becoming immortalized by telomerase alone. As human keratinocytes age in culture, protein levels of the tumor suppressor p16INK4a continue to increase, resulting in growth arrest independent of telomere length. Differences in culture conditions have been shown to modulate both p16INK4a expression and replicative capacity of human keratinocytes; however, the mechanism of p16INK4a induction under these conditions is unknown. Using multiple primary keratinocyte cell strains, we verified a delay in p16INK4a induction and an extended lifespan of human keratinocytes when grown in co-culture with post-mitotic fibroblast feeder cells as compared with keratinocytes grown on tissue culture plastic alone. Evaluation of gene expression levels in the two culture conditions by microarray analysis, and subsequent validation, demonstrated that keratinocytes cultured on plastic alone had significantly increased expression of many genes involved in keratinocyte migration and reduced expression levels of genes involved in keratinocyte differentiation. Higher levels of p16INK4a expression were present in cells that also displayed increased amounts of autophosphorylated focal adhesion kinase and urokinase plaminogen activator receptor (uPAR), both markers of keratinocyte migration. Furthermore, when tyrosine phosphorylation or urokinase-type plasminogen activator (uPA)/uPAR function was inhibited, both keratinocyte migration and p16INK4a expression were reduced. Our results indicate that keratinocytes cultured in the absence of feeder cells exhibit a migratory phenotype and suggest that p16INK4a is selectively induced under these conditions by a mechanism involving tyrosine kinase activity and the urokinase plasminogen activation system.

Energetic stress in combination with tumor necrosis factor-alpha (TNF-alpha) induces apoptosis of human fibroblasts (WI-38) in vitro: reduced responsiveness of senescent cells.

The NaCl content of the culture medium of human fibroblasts (WI-38) was elevated from 0.12 M to 0.23 M. Previously it had been shown that exposure of fibroblasts to such high NaCl conditions resulted in a rise of both glycolysis and respiration ("energetic stress"), mainly due to increased demands of energy for active ion transport ("sodium pump"). While "young" (Phase II) and "senescent" (Phase III) cells did not show a significant increase of apoptosis over the basal rate (ca. 4%) under treatment with either a high-NaCl medium or TNF-alpha (10 nM) alone, combined treatment resulted in a strong increase in Phase II cells and a significantly lesser rise in the case of "senescent" Phase III cells. We conclude, therefore, that energetic stress stimulates sensitivity to apoptosis by (in the presence of) TNF-alpha, especially pronounced in potentially replicating "young" as compared with irreversible postmitotic ("senescent") fibroblasts. Possible causes of this differential responsiveness and implications for the in vivo situation (in the organism) were discussed.

Isolation and Identification of Psoralen plus Ultraviolet A (PUVA)-Induced Genes in Human Dermal Fibroblasts by Polymerase Chain Reaction-Based Subtractive Hybridization

Premature aging of the skin is a prominent side-effect of psoralen photoactivation, a therapy used for a variety of skin disorders. Recently, we demonstrated that treatment of human dermal fibroblasts with 8-methoxypsoralen and ultraviolet A irradiation resulted in a permanent growth arrest with a switch of mitotic to postmitotic fibroblasts. Furthermore, an upregulation of matrix-degrading metalloproteinases and a high level of de novo expression of the senescence-associated beta-galactosidase was detected in the PUVA-treated postmitotic fibroblasts. The molecular basis for this PUVA-induced change in the functional and morphologic phenotype of fibroblasts resembling or mimicking replicative senescence is, however, unknown. Herein after, we have used a polymerase chain reaction-based subtractive hybridization protocol to identify human genes that are induced by PUVA treatment. Application of polymerase chain reaction-Select resulted in the cloning of four PUVA genes. Sequence analysis and homology searches identified three cDNA clones of known genes related to cell cycle regulation (p21waf1/cip1), stress response (ferritin H) and connective tissue metabolism (tissue inhibitor of metalloproteinases-3), whereas one cDNA clone represented a novel gene (no. 478). Northern blot analyses were performed to confirm a PUVA-dependent increase in specific mRNA levels in human dermal fibroblasts in vitro. This report on the identification of growth arrest related genes in PUVA-treated fibroblasts may stimulate further research addressing the causal role of these known and novel genes in extrinsic and intrinsic aging processes on a molecular and cellular level.

Keratinocyte Growth Regulation in Defined Organotypic Cultures Through IL-1-Induced Keratinocyte Growth Factor Expression in Resting Fibroblasts

Balanced keratinocyte proliferation and differentiation resulting in regular tissue organization strictly depend on dermal support. Organotypic cultures represent biologically relevant in vitro models to study the molecular mechanism of the underlying dermal-epidermal interactions. To mimic the state of resting fibroblasts in the dermis, postmitotic (irradiated) fibroblasts were incorporated in the collagen matrix, where they typically support epidermal proliferation and tissue organization. In coculture with keratinocytes, fibroblasts exhibit an enhanced expression of keratinocyte growth factor and the interleukin-1 receptor (type I), which further increase with culture time. In cocultured keratinocytes, keratinocyte growth factor receptor as well as RNA expression and protein release of interleukin-1alpha and interleukin-1beta are upregulated. We hypothesized that the modulated cytokine expression represents a basic mechanism for keratinocyte growth regulation. The functional significance of this double paracrine pathway, i.e., induction of keratinocyte growth factor expression in fibroblasts by keratinocytes via release of interleukin-1, was confirmed by interfering with both signaling elements: (i) interleukin-1-neutralizing antibodies and interleukin-1 receptor antagonist significantly inhibited keratinocyte growth factor release, keratinocyte proliferation, and tissue formation comparable to the effect produced by keratinocyte-growth-factor-blocking antibodies; (ii) addition of keratinocyte growth factor to cocultures with inactivated interleukin-1 pathway completely reverted growth inhibition; (iii) in organotypic cocultures with subthreshold fibroblast numbers both interleukin-1 and keratinocyte growth factor restored the impaired epidermal morphogenesis. Thus, epidermal tissue regeneration in organotypic cocultures is mainly regulated by keratinocyte-derived interleukin-1 signaling, which induces keratinocyte growth factor expression in cocultured fibroblasts. This demonstrates a novel role for interleukin-1 in skin homeostasis substantiating data from wound healing studies in vivo.

The Small Proteoglycan Fibromodulin Is Expressed in Mitotic, but Not in Postmitotic Fibroblasts

The small proteoglycan fibromodulin is involved in a variety of adhesion processes of connective tissue, binds to collagen and seems to influence fibrillogenesis by regulating collagen fibril spacing and thickness. Using subtractive hybridisation and Northern blotting, we found expression of the fibromodulin gene only in mitotic but not in mitomycinC-induced postmitotic fibroblasts. Furthermore, we could show that in skin only fibroblasts but not endothelial cells and keratinocytes express fibromodulin. With age the thickness of collagen fibrils increases as well as the occurrence of postmitotic fibroblasts. We could show with different chemotaxis and similar collagen gel contraction experiments further physiological differences between mitotic and postmitotic fibroblasts. This is the first report showing the switch of mitotic to postmitotic fibroblasts at the molecular level. Therefore, fibromodulin provides a specific marker for mitotic versus postmitotic fibroblasts and could indicate cell ageing. Mitomycin C is therapeutically used in bladder carcinoma and could thus induce premature ageing of fibroblasts.

Psoralen photoactivation promotes morphological and functional changes in fibroblasts in vitro reminiscent of cellular senescence.

Premature aging of the skin is a prominent side effect of psoralen photoactivation, a treatment used widely for various skin disorders. The molecular mechanisms underlying premature aging upon psoralen photoactivation are as yet unknown. Here we show that treatment of fibroblasts with 8-methoxypsoralen (8-MOP) and subsequent ultraviolet A (UVA) irradiation resulted in a permanent switch of mitotic to stably postmitotic fibroblasts which acquired a high level of de novo expression of SA-beta-galactosidase, a marker for fibroblast senescence in vitro and in vivo. A single exposure of fibroblasts to 8-MOP/UVA resulted in a 5.8-fold up-regulation of two matrix-degrading enzymes, interstitial collagenase (MMP-1) and stromelysin-1 (MMP-3), over a period of >120 days, while TIMP-1, the major inhibitor of MMP-1 and MMP-3, was only slightly induced. This imbalance between matrix-degrading metalloproteases and their inhibitor may lead to connective tissue damage, a hallmark of premature aging. Superoxide anion and hydrogen peroxide, but not singlet oxygen, were identified as important intermediates in the downstream signaling pathway leading to these complex fibroblast responses upon psoralen photoactivation. Collectively, the end phenotype induced upon psoralen photoactivation shares several criteria of senescent cells. In the absence of detailed molecular data on what constitutes normal aging, it is difficult to decide whether the changes reported here reflect mechanisms underlying normal cellular aging/senescence or rather produce a mimic of cellular aging/senescence by quite different pathways.

Glutathione response after UVA irradiation in mitotic and postmitotic human skin fibroblasts and keratinocytes.

Since Hayflick's pioneering work in the early sixties, human diploid fibroblasts have become a widely accepted in vitro model system. Recently, Bayreuther and co-workers extended this experimental approach showing that fibroblasts in culture resemble, in their design, the hemopoietic stem-cell differentiation system. They found that the chemical agent mitomycin C accelerates the differentiation pathway from mitotic to postmitotic fibroblasts. We measured the response of endogenous glutathione levels after UVA irradiation (320-400 nm) in mitotic and mitomycin C-induced postmitotic human skin fibroblasts and foreskin-derived keratinocytes. The initial levels in mitotic foreskin derived human fibroblasts were 14.4 nmol glutathione per mg protein, whereas a 30% higher value was obtained in matching foreskin-derived keratinocytes. Similar elevated levels of this important intracellular free radical scavenging system were found in fibroblasts of a donor suffering from xeroderma pigmentosum. Furthermore, three to four times higher levels of glutathione in mitomycin C-treated mitotic fibroblasts have been determined. In mitotic skin fibroblasts, UVA irradiation resulted in a depletion of glutathione up to 90% following a fluence of 1.0 MJ/m2 UVA radiation. Higher initial glutathione levels were found in keratinocytes and mitomycin C-treated skin fibroblasts. In these fibroblasts a 70% depletion was detected and a much lower depletion (10-20%) was seen in some keratinocyte cell lines following fluences up to 1.0 MJ/m2. The depletion in skin fibroblasts was retained after 24 h following a fluence of 0.75 MJ/m2 UVA light. In view of the fact that glutathione has been shown to be involved in a variety of metabolic processes and plays a role in cellular protection against UVA radiation, our results imply that the fibroblast differentiation system is a very useful tool to unravel the complex mechanism of UVA-induced oxidative stress.

DNA Synthesis and Fos and Jun Protein Expression in Mitotic and Postmitotic WI-38 Fibroblasts in Vitro

Normal human embryonic lung fibroblasts WI-38 differentiate spontaneously along the cell lineage mitotic fibroblasts (MF) I, II, and III and postmitotic fibroblasts (PMF) IV, V, VI, and VII in the fibroblast stem cell system in vitro, when appropriate methods are applied. The mitotic fibroblasts can be induced to shift to postmitotic fibroblasts by two treatments with mitomycin C (2× MMC) in a short period of time compared to spontaneous development. Mitotic and postmitotic fibroblast cell types have specific morphological and biochemical properties, e.g., [35S]methionine polypeptide markers in 2D PAGE. Spontaneously arisen and experimentally induced (2× MMC) PMF have the same morphological and biochemical characteristics. Mitotic fibroblasts have 2n DNA and undergo DNA synthesis for reduplication. Postmitotic cells undergo, on average, two rounds of DNA synthesis for endoreduplication (polyploidization). Spontaneously arisen and experimentally induced postmitotic populations are composed of postmitotic fibroblasts PMF IV, V, and VI with 2n, 4n, and 8n DNA. DNA synthesis of mitotic and postmitotic WI-38 cell populations may be regulated by the expression of Fos and Jun proteins. The Fos level of MFs was higher by a factor of 15-24 and the Jun level of MFs by a factor of 4.2-6.3 than those of spontaneously arisen PMFs. In 2× MMC-induced PMFs, the Fos level was about 4.4-7.5 times higher and the Jun level 1.7-3.3 times higher than that of spontaneously arisen PMFs. The down-regulation of these two parameters is a normal event in the development of mitotic to postmitotic WI-38 fibroblasts in the fibroblast stem cell system and is not related to cellular aging.

Aphidicolin inhibits excision repair of UV-induced pyrimidine photodimers in low serum cultures of mitotic and mitomycin C-induced postmitotic human skin fibroblasts

The rates of formation and excision of UVC light-induced cyclobutane-type pyrimidine photodimers were determined in cultures of foreskin-derived normal human fibroblasts in mitotic (MF) and mitomycin-C (MMC)-induced postmitotic fibroblasts (PMF). Characteristic morphological changes support the notion that MMC accelerates the differentiation pathway from MF to PMF. In cultures treated with aphidicolin, I am able to show that this inhibitor of alpha and/or delta polymerases significantly inhibits the repair of pyrimidine photodimers in foreskin-derived mitotic and MMC-induced postmitotic fibroblasts in low serum cultures (0.5%) following UVC irradiation. Over the concentration range of 0-2 micrograms/ml of aphidicolin, there is a strong concentration-dependent inhibition of repair in cells treated with 10 J/m2 of UVC and incubated with aphidicolin during the post-incubation time (0-24 h). The results demonstrate that pyrimidine photodimers are repaired in low serum cultures by an alpha- and/or delta-polymerase-dependent pathway. These data also imply that the fibroblast differentiation system is a very useful tool to unravel the complex mechanisms of UV-induced DNA damage and repair.

Stimulation of cell proliferation by hyaluronidase during in vitro aging of human skin fibroblasts

The effect of the degradation of extracellular hyaluronan on the proliferation of human skin fibroblasts in serial cultures during in vitro aging was investigated. Human skin fibroblasts at different time intervals from 3rd to 36th passages were exposed after plating to bovine testicular hyaluronidase. The enzyme treatment resulted in an increase in cell proliferation (cell number vs. time) as compared to the untreated control fibroblasts. The effect was dose dependent, reversible, and was independent of the type of the glycosidic linkage cleaved in hyaluronan. The increased proliferation was observed at all passages when untreated cells underwent mitosis. The degradation of hyaluronan induced cell proliferation up to the presenescent phase. Depletion of hyaluronan did not induce proliferation of postmitotic fibroblasts. The incorporation of 3H-glucosamine into hyaluronan decreased with increasing cell passages (increase of the number of population doublings). Twenty-fourth passage fibroblasts accumulated about two time less hyaluronan in the medium than ninth passage cultures. Following hyaluronidase treatment, the amount of newly synthesized, labeled hyaluronan increased in the medium. Accordingly, the fibroblasts restored the degraded hyaluronan even in the declining phase of proliferation (phase III according to Hayflick).

May ultraviolet light-induced ornithine decarboxylase response in mitotic and postmitotic human skin fibroblasts serve as a marker of aging and differentiation?

Since Hayflick’s pioneering work in the early sixties, human diploid fibroblasts have become a widely accepted in vitro model system for gerontological research. Most recently, Bayreuther and co-workers extended this experimental approach showing that fibroblasts in culture resemble in their design the hemopoietic stem-cell differentiation system. Using this model, we have found in human skin fibroblasts following mitomycin-C (MMC) treatment characteristic morphological changes of the fibroblasts and specific shifts in the [35S]methionine polypeptide pattern of the total cellular proteins which support the notion that MMC accelerates the differentiation pathway from mitotic (MF) to postmitotic fibroblasts (PMF). Ornithine decarboxylase (ODC) can be activated by ultraviolet light (UV) and is involved in the synthesis of polyamines which play a role in the regulation of DNA synthesis and cell proliferation. Therefore, ODC may participate in the modulation of gene expression. ODC may even serve as a biochemical marker of the mutagenic and carcinogenic effects of ultraviolet light; therefore, we tested this interesting enzyme in the fibroblast differentiation system. Indeed, we were able to show that UV-induced ODC response is significantly reduced in the MMC-induced postmitotic stage of fibroblasts originally derived from a 9-year-old female. We compared this finding with previous results from our laboratory, where we have demonstrated that ODC in human skin fibroblasts from younger donors can be significantly more stimulated by UV compared to the enzyme activities in fibroblasts from older donors. We conclude that UV-induced ODC response may serve as a marker of differentiation and aging. Our results also imply that the fibroblast differentiation system is a very useful tool to unravel the complex mechanisms of skin aging.

Differentiation of primary and secondary fibroblasts in cell culture systems

As a function of the advancing development of Valo chicken, C3H mice, BN rats, and man in the embryonic, juvenile, adolescent, and senescent phases, stem cells and fibroblasts in the connective tissues of skin and lung differentiate along an 11-stage differentiation sequence in five compartments of the fibroblast stem cell system, when studied in primary ex vivo-in vitro systems. In the fibroblast stem cell system, three stem cells develop in the stem cell compartment along the cell lineage S1-S2-S3, three mitotic fibroblasts (MF) differentiate along the sequence MF I-MF II-MF III in the fibroblast progenitor compartment, three postmitotic fibroblasts (PMF) proceed in the fibroblast maturing compartment along the row PMF IV-PMF V-PMF VI. PMF VI is the terminally differentiated end cell of the fibroblast stem cell system. After a species- and tissue-specific period of high metabolic activity, PMF VI either dies as PMF VIIa in the fibroblast apoptosis compartment or transforms as PMF VIIb in the fibroblast transforming compartment. The reiterated appearance of the 11 cell types in primary stem cell and fibroblast populations and the reiterated age-related changes in the cell type composition of the primary stem cell and fibroblast populations make it very likely that stem cell, mitotic and postmitotic fibroblast equivalents exist in vivo and that age-related changes of the frequencies of the stem cell and fibroblast equivalents result from the progressing differentiation of stem cell, mitotic, and postmitotic fibroblast equivalents along the 11 stage differentiation sequence in the fibroblast equivalent stem cell system in vivo. Secondary fibroblast populations derived from connective tissue of prenatal and postnatal skin of Valo chicken, C3H mice, BN rats, and man, including the normal embryonic human lung fibroblast cell line WI38, were also found to develop along a terminal stem cell sequence. Thus, secondary fibroblast populations in vitro constitute a representative material for studies of general and special issues of cell biology, such as terminal differentiation, aging, apoptosis, and transformation, as long as stem cell system-specific concepts and methods are employed in such investigations.

Postmitotic human dermal fibroblasts preserve intact feeder properties for epithelial cell growth after long-term cryopreservation

In vitro, human dermal fibroblasts (HDF) differentiate through morphologically and biochemically identified compartments. In the course of this spontaneous differentiation through mitotic and postmitotic states, a tremendous increase in cellular and nuclear size occurs. Induction of postmitotic states can be accelerated by chemical (e.g., mitomycin C) or physical (e.g., x-ray) treatments. Such experimentally induced postmitotic HDF cells support very efficiently the growth of cutaneous epithelial cells, i.e. interfollicular keratinocytes and follicular outer root sheath cells, especially in primary cultures starting from very low cell seeding densities. The HDF feeder system provides more fundamental and also practical advantages, i.e. use of initially diploid human fibroblasts from known anatomic locations, easy handling and excellent reproducibility, and the possibility of long-term storage by incubation at 37 degrees C. Conditions for the cryogenic storage of postmitotic HDF cells in liquid nitrogen are presented and related to the feeder capacity for epithelial cell growth. Because postmitotic HDF cells preserve intact feeder properties after long-term storage, the immediate availability of feeder cells and the possibility to repeat experiments with identical materials further substantiate the usefulness of this feeder system.

Differential degradation of intracellular proteins in human skin fibroblasts of mitotic and mitomycin-C (MMC)-induced postmitotic differentiation states in vitro

Abstract Rates of degradation of short- and long- lived proteins were analysed in homogeneous fibroblast cultures of mitotic or mitomycin C (MMC)-induced postmitotic states. When the highly mitotic MFII type cells - the major cell type of so called “early passage” or “young” fibroblasts - differentiate into MFIII type cells, the last mitotic fibroblast type, and further into MMC-induced postmitotic fibroblasts, the degradation of short-lived proteins increases by a factor of 1.4, resulting in significantly reduced halflives of these proteins in the postmitotic fibroblasts. From the highly mitotic MFII to the final postmitotic PMFVI-type cells via the intermediates MFIII, PMFIV and PMFV, the half lives ( t 1/2) of short-lived proteins decrease by a total of 122 min in average, from 362 to 240 min. Degradation of long-lived proteins did not change significantly from cell type MFII to PMFVI. As analysed by two-dimensional (2D)-gel electrophoresis the half-lives of the mitotic and postmitotic cell-type-specific proteins except one, protein PIVa (33 kDa; Pi 5.0), range between 33.2 h and 62.9 h. Protein PIVa, the first protein specific for postmitotic cells, is initially expressed 18 h after the induction of the postmitotic state by mitomycin C (MMC) and has a half-life of approximately 66 min. This may indicate that protein PIVa could function as one possible regulatory factor controlling the postmitotic differentiation state.

Mitomycin C-induced postmitotic fibroblasts retain the capacity to repair pyrimidine photodimers formed after UV-irradiation

The formation and excision of UV-C light-induced cyclobutane-type pyrimidine photodimers were determined in cultures of human skin fibroblasts at time zero and several weeks following treatment with mitomycin C (MMC). Characteristic morphological changes of the fibroblasts and specific shifts in the [35S]methionine polypeptide pattern of total cellular proteins support the notion that MMC accelerates the differentiation pathway from mitotic (MF) to post-mitotic fibroblasts (PMF). No discernible difference could be detected between the fluence-response curves of pyrimidine dimers for untreated and MMC-treated repair-deficient xeroderma pigmentosum cells of group A. Furthermore we investigated the removal of pyrimidine dimers in 3 normal human skin fibroblast strains frequently used in mutation, transformation and aging research. We were able to demonstrate that no significant difference exists in the rate and extent of the excision-repair response to thymine-containing pyrimidine dimers following UV-irradiation shortly after MMC treatment of fibroblasts and in the MMC-induced PMF stage of these cells.

Selective enrichment and biochemical characterization of seven human skin fibroblasts cell types in vitro

The mitotic and postmitotic populations of the human skin fibroblast cell line HH-8 are heterogeneous when studied in vitro. There are reproducible changes in the frequencies of the mitotic fibroblasts (MF), MF I, MF II, MF III, and the postmitotic fibroblasts (PMF), PMF IV, PMF V, PMF VI, and PMF VII. For biochemical characterization, methods for selective enrichment of homogeneous populations of these seven fibroblast cell types have been established. Clonal populations with 95% purity for the mitotic fibroblasts MF I, MF II, and MF III can be raised in uniform clone types of fibroblasts (CTF) CTF I, CTF II, and CTF III. Pure clonal subpopulations of MF I type cells are present in mass populations in the range of 1–20 cumulative population doublings (CPD). Populations of mitotic fibroblasts represent nearly homogeneous populations of MF II (75–85 % purity) in the range of 28–34 CPD and MF III (73–86% purity) in the range of 48–53 CPD. These populations can be easily expanded to up to 10 7–10 8 cells. The spontaneous transition of MF III to PMF VI takes 140–180 days. In order to shorten this period and increase the proportion of distinct postmitotic types, mitotic fibroblast mass populations (CPD 30–32, MF II: 75–85 % purity) have been induced by uv-irradiation to differentiate to nearly homogeneous populations of PMF IV, PMF V, PMF VI, and PMF VII within 4 to 36 days of culture. Using this method, 10 7 cells of one differentiation stage can be obtained. Spontaneously arising and experimentally selected or induced homogeneous clonal and mass populations of MF I, MF II, MF III, PMF IV, PMF V, PMF VI, and PMF VII express an identical differentiation-dependent and cell-type-specific [ 35S]methionine-labeled polypeptide pattern.