Frontiers in crop production: chemical research objectives.

The science of chemistry and chemicals will continue to be an integral part of future crop production technologies. In assessing and defining the future role of chemistry three imperatives must be considered: (i) the necessity to preserve natural resources, (ii) the complementary solutions offered by the rapidly advancing biological sciences, and (iii) the specific requirements of developing regions where increasing crop productivity is most important. Chemical research objectives for improving crop protection and crop growth must take into account the perceivable and potential changes in crop production techniques, which, in turn, are dictated by a number of accentuating constraints.

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.

Adaptive antioxidant response of manganese-superoxide dismutase following repetitive UVA irradiation.

In response to the attack of reactive oxygen species, the skin has developed a complex antioxidant defense system including among others the manganese-superoxide dismutase (MnSOD). MnSOD dismutates the superoxide anion (O2*-) derived from the reduction of molecular oxygen to hydrogen peroxide (H2O2), which is detoxified by glutathione peroxidase to water and molecular oxygen. We have addressed the question whether MnSOD is inducible upon UVA irradiation and whether repetitive UV exposure, as practiced for the light-hardening during phototherapy of various photodermatoses, can even enhance the adaptive antioxidant response. Single exposure of four different strains of fibroblasts to UVA irradiation resulted in a dose- and time-dependent increase in specific MnSOD mRNA levels. Interestingly, repetitive UVA exposure at days 1, 2, and 3 at a dose rate of 200 kJ per m2 resulted in a 5-fold induction of specific MnSOD mRNA levels following the third UVA exposure. Similar results were obtained for MnSOD activity. This adaptive response in terms of upregulation of the antioxidant enzyme MnSOD correlates with the protection against high UV doses, if cells were preexposed to sublethal UV doses. Importantly, MnSOD substantially differed between the tested individuals in both mRNA and activity levels. Taken together, we here provide evidence for the increasing induction of MnSOD upon repetitive UVA irradiation that may contribute to the effective adaptive UVA response of the skin during light hardening in phototherapy. Interindividual differences in the inducibility of MnSOD might account for differences in the susceptibility to develop photodermatologic disorders related to photosensitivity, photoaging, and skin cancer. The molecular basis for interindividual differences in the inducibility of antioxidant enzymes remains to be elucidated.

Selective pick-up of increased iron by deferoxamine-coupled cellulose abrogates the iron-driven induction of matrix-degrading metalloproteinase 1 and lipid peroxidation in human dermal fibroblasts in vitro: a new dressing concept.

Using atomic absorption spectrum analysis, we found iron levels in exudates from chronic wounds to be significantly increased (3.71 +/- 1.56 micromol per g protein) compared to wound fluids from acute wounds derived from blister fluids (1.15 +/- 0.62 micromol per g protein, p < 0.02), drainage fluids of acute wounds (0.87 +/- 0.34 micromol per g protein, p < 0.002), and pooled human plasma of 50 volunteers (0.42 micromol per g protein). Increased free iron and an increase in reactive oxygen species released from neutrophils represent pathogenic key steps that --via the Fenton reaction - are thought to be responsible for the persistent inflammation, increased connective tissue degradation, and lipid peroxidation contributing to the prooxidant hostile microenvironment of chronic venous leg ulcers. We herein designed a selective pick-up dressing for iron ions by covalently binding deferoxamine to cellulose. No leakage occurred following gamma sterilization of the dressing and, more importantly, the deferoxamine-coupled cellulose dressing retained its iron complexing properties sufficient to reduce iron levels found in chronic venous ulcers to levels comparable to those found in acute wounds. In order to study the functionality of the dressing, human dermal fibroblasts were exposed to a Fenton reaction mimicking combination of 220 microM Fe(III) citrate and 1 mM ascorbate resulting in a 4-fold induction of matrix-degrading metalloproteinase 1 as determined by a matrix-degrading metalloproteinase 1 specific enzyme-linked immunosorbent assay. This induction was completely suppressed by dissolved deferoxamine at a concentration of 220 microM or by an equimolar amount of deferoxamine immobilized to cellulose. In addition, the Fe(III) citrate and ascorbate driven Fenton reaction resulted in an 8-fold increase in malondialdehyde, the major product of lipid peroxidation, as determined by high pressure liquid chromatography. This increase in malondialdehyde levels could be significantly reduced in the presence of the selective pick-up dressing coupled with deferoxamine suggesting that the deferoxamine dressing, in fact, prevents the development of a damaging prooxidant microenvironment and also protects from unfavorable consequences like matrix-degrading metalloproteinase 1 and lipid peroxide induction.

Hydrogen peroxide (H2O2) increases the steady-state mRNA levels of collagenase/MMP-1 in human dermal fibroblasts.

Reactive oxygen species (ROS) have been shown to be important messenger molecules in the induction of several genes. In human dermal fibroblasts the herbicide paraquat (PQ2+) was used to induce intracellular oxidative stress that was modulated by the inhibition of copper, zinc superoxide dismutase (Cu,ZnSOD), glutathione peroxidase (GSHPx), catalase, and blocking of the Fenton reaction. Interstitial collagenase (MMP-1) mRNA increased time dependently for up to 72 h following paraquat treatment. A correlation with the translation of MMP-1 could, however, only be detected up to 24 h, indicating an uncoupling of transcription and translation. Interleukin-1 alpha and beta mRNA showed two peaks at 6 h and 72 h. The inhibition of catalase by aminotriazol (ATZ), inhibition of GSHPx by buthionine sulfoximine (BSO), and blocking the Fenton reaction by the iron chelator desferrioxamine (DFO) in concert led to an increase in steady-state MMP-1 mRNA levels, possibly dependent on intracellular H2O2 increase. This combined treatment potentiated MMP-1 mRNA induction up to 6.5-fold compared to paraquat treated controls. Furthermore, exogenously added H2O2 caused an increase in MMP-1 mRNA levels. In contrast, inhibition of Cu,ZnSOD by diethyldithiocarbamate (DDC), leading to diminished H2O2 production from O2.-, decreased MMP-1 mRNA induction. Collectively, our data provide evidence that H2O2 is an important intermediate in the downstream signalling pathway finally leading to the induction of increased steady state MMP-1 mRNA levels. The synthesis of MMPs may contribute to connective tissue damage in vivo related to photoaging, inflammatory diseases, and tumor invasion.

The first peak of the UVB irradiation-dependent biphasic induction of vascular endothelial growth factor (VEGF) is due to phosphorylation of the epidermal growth factor receptor and independent of autocrine transforming growth factor alpha.

Ultraviolet B (UVB) irradiation, the major damaging component of sunlight, has earlier been reported to enhance cutaneous angiogenesis in chronically sun-exposed skin. We herein provide first evidence for a biphasic induction of the vascular endothelial growth factor (VEGF) following UVB irradiation of the human epidermal cell line HaCaT. The first VEGF peak occurred on mRNA level at 1 h and on protein level at 4 h postirradiation and is fully mediated by the UVB-dependent phosphorylation of the epidermal growth factor receptor, which subsequent to its phosphorylation also initiates at least in part the synthesis of transforming growth factor alpha that confers as shown previously the second late VEGF peak at 8 h on mRNA and at 24 h on protein level.

Singlet oxygen is an early intermediate in cytokine-dependent ultraviolet-A induction of interstitial collagenase in human dermal fibroblasts in vitro.

Ultraviolet (UV) A irradiation of human dermal fibroblasts elicits an increase in specific mRNA amounts and bioactivities of the cytokines IL-1alpha, IL-1beta, and IL-6. These effects are enhanced in deuterium oxide-based medium and are diminished in the presence of non-toxic concentrations of sodium azide. Furthermore, generating singlet oxygen outside the cells by irradiation of rose bengal-coated resin particles with visible light (lambda > 450 nm) results in the induction of interstitial collagenase, IL-1 and IL-6, similar to the response observed with UVA irradiation. These observations suggest that singlet oxygen is an early intermediate in the signaling pathway of IL-1 and IL-6 mediating UVA induction of interstitial collagenase (E.C. 3.4.24.7). Furthermore, singlet oxygen appears to initiate this complex UV response at the cell membrane.

Adaptive antioxidant response protects dermal fibroblasts from UVA-induced phototoxicity.

In response to the attack of reactive oxygen species (ROS) produced upon UV irradiation, the skin has developed a complex antioxidant defense system. Here we report that, in addition to the previously published induction of manganese superoxide dismutase (MnSOD) activity, single and, to a higher extent, repetitive low-dose UVA irradiation also leads to a substantial upregulation of glutathione peroxidase (GPx) activity. This concomitant adaptive response of two antioxidant enzymes acting in the same detoxification pathway coincided with the protection from high-UVA-dose-induced cytotoxicity conferred by low-dose UVA preirradiation. Whereas an interval of 24 h did not, an interval of 12 h did lead to the induction of MnSOD activity and, under selenium-supplemented conditions, of GPx activity as well, conferring definite cellular protection from UVA-induced phototoxicity. Moreover, under selenium-deficient conditions, which abrogate the UVA-mediated induction of GPx activity, adaptive protection against the cytotoxic effects of high UVA doses was significantly lower compared with selenium supplementation. Isolated 4.6-fold overexpression of MnSOD activity in stably transfected fibroblasts led to specific resistance from UVA-mediated phototoxicity under selenium-deficient conditions. Collectively, these data indicate that the concomitant induction of MnSOD and GPx activity is related to the optimal adaptive protection from photooxidative damage. This adaptive antioxidant protection clearly depends on the irradiation interval and a sufficient selenium concentration, findings that may have important implications for the improvement of photoprotective and phototherapeutic strategies in medicine.

Ultraviolet-B induction of interstitial collagenase and stromelyin-1 occurs in human dermal fibroblasts via an autocrine interleukin-6-dependent loop.

Ultraviolet-B irradiation of human dermal fibroblasts has earlier been shown to induce matrix-degrading metalloproteinases, thus driving connective tissue degradation in photoaging and photocarcinogenesis. Herein, we report that Ultraviolet-B irradiation led to a dramatic increase in specific mRNA and protein levels of interstitial collagenase, stromelysin and interleukin-6. By contrast, the major tissue inhibitor of matrix-degrading metalloproteinases, TIMP-1, was unaffected. Monospecific neutralizing antibodies directed against human interleukin-6 significantly reduced the interstitial collagenase and stromelysin-1 protein levels. Taken together, our data provide the first evidence that Ultraviolet-B induction of interstitial collagenase and stromelysin-1 occurs via the synthesis and release of interleukin-6. Hence, this newly identified autocrine mechanism may contribute to dermal photodamage.

PCR-based subtractive hybridization identifies repressed genes in growth-arrested human dermal fibroblasts following combined treatment with 8-methoxypsoralen and UVA irradiation (PUVA).

To identify genes which are repressed in growth-arrested human dermal fibroblasts upon a single treatment with 8-methoxypsoralen and UVA irradiation (PUVA) we have used a PCR-based subtractive hybridization protocol resulting in cloning of four PUVA-repressed genes. Sequence analysis and homology searches identified three known genes related to growth control, lipid and connective tissue metabolism. One cDNA clone represented a novel gene. Northern blot analyses confirmed a PUVA-dependent reduction in mRNA expression in fibroblasts in vitro. The identification of growth arrest related repressed genes in PUVA-treated fibroblasts may stimulate further research addressing the causal role of these genes in the control and regulation of the postmitotic phenotype of fibroblasts on a molecular and cellular level.

Photoaging of the skin from phenotype to mechanisms.

The skin is increasingly exposed to ambient UV-irradiation thus increasing its risk for photooxidative damage with longterm detrimental effects like photoaging, which is characterized by wrinkles, loss of skin tone, and resilience. Photoaged skin displays prominent alterations in the cellular component and the extracellular matrix of the connective tissue with an accumulation of disorganized elastin and its microfibrillar component fibrillin in the deep dermis and a severe loss of interstitial collagens, the major structural proteins of the dermal connective tissue. The unifying pathogenic agents for these changes are UV-generated reactive oxygen species (ROS) that deplete and damage non-enzymatic and enzymatic antioxidant defense systems of the skin. As well as causing permanent genetic changes, ROS activate cytoplasmic signal transduction pathways in resident fibroblasts that are related to growth, differentiation, senescence, and connective tissue degradation. This review focuses on the role of UV-induced ROS in the photodamage of the skin resulting in biochemical and clinical characteristics of photoaging. In addition, the relationship of photoaging to intrinsic aging of the skin will be discussed. A decrease in the overall ROS load by efficient sunscreens or other protective agents may represent promising strategies to prevent or at least minimize ROS induced photoaging.

A newly adapted pulsed-field gel electrophoresis technique allows to detect distinct types of DNA damage at low frequencies in human dermal fibroblasts upon exposure to non-toxic H2O2 concentrations.

Reactive oxygen species (ROS) comprise several oxygen containing compounds, among them hydrogen peroxide (H2O2), which are generated by internal and external sources and play pleiotropic roles in physiological and pathological states. Skin cells as well as cells from other tissues have developed antioxidant defense mechanisms to protect themselves from high concentrations of ROS. Although biological and pathological roles of ROS have previously been elucidated, so far only limited knowledge exists regarding ROS-mediated generation of DNA breaks and base lesions occurring at low frequency in intact skin cells. This study was therefore designed to probe a newly adapted pulsed-field gel electrophoresis technique for the adequate measurement of high molecular weight DNA fragments as well as to investigate the protective role of the antioxidant enzyme catalase against H2O2-mediated damage in human dermal fibroblasts. We stably transfected and overexpressed the full-length catalase cDNA in the human dermal fibroblast cell line 1306 in culture and found that these cells are significantly more protected from cytotoxicity, overall DNA strand breaks, and 8-oxodeoxyguanine base lesions resulting from H2O2-triggered oxidative stress compared to vector-transfected 1306 cells or secondary dermal fibroblasts. This work has outlined the importance of catalase in the protection from H2O2-mediated cytotoxicity and DNA damage which--if unbalanced--even when occurring at low frequency are known to lead to genomic instability, a hallmark in carcinogenesis and premature aging.

Ultraviolet B wavelength dependence for the regulation of two major matrix-metalloproteinases and their inhibitor TIMP-1 in human dermal fibroblasts.

The wavelength dependence for the regulation of two major matrix-metalloproteinases, interstitial collagenase (MMP-1) and stromelysin-1 (MMP-3), and their major inhibitor, tissue inhibitor of metalloproteinases (TIMP-1), was studied in human dermal fibroblasts in vitro. Monochromatic irradiation at 302, 307, 312 and 317 nm with intensities ranging from 20 to 300 J/m2 increased MMP-1 and MMP-3 mRNA steady-state levels and the secretion of the corresponding proteins up to 4.4-fold, whereas almost no increase was observed at wavelengths < 290 nm. In contrast, the synthesis of TIMP-1 increased only marginally. This imbalance may contribute to the severe connective tissue damage related to photoaging of the skin. The wavelengths responsible for MMP-1 and MMP-3 induction reported here are distinct from the absorption spectrum of DNA and are different from results previously reported in the literature. Importantly, they overlap with wavelengths whose intensity is predicted to increase on the earth's surface upon ozone depletion. Intensities and particular wavelengths used in our studies in vitro can be absorbed readily by fibroblasts within the skin in vivo and, thus, are relevant for risk assessment and development of protective agents.

Ultraviolet B wavelength dependence for the regulation of two major matrix-metalloproteinases and their inhibitor TIMP-1 in human dermal fibroblasts.

The wavelength dependence for the regulation of two major matrix-metalloproteinases, interstitial collagenase (MMP-1) and stromelysin-1 (MMP-3), and their major inhibitor, tissue inhibitor of metalloproteinases (TIMP-1), was studied in human dermal fibroblasts in vitro. Monochromatic irradiation at 302, 307, 312 and 317 nm with intensities ranging from 20 to 300 J/m2 increased MMP-1 and MMP-3 mRNA steady-state levels and the secretion of the corresponding proteins up to 4.4-fold, whereas almost no increase was observed at wavelengths < 290 nm. In contrast, the synthesis of TIMP-1 increased only marginally. This imbalance may contribute to the severe connective tissue damage related to photoaging of the skin. The wavelengths responsible for MMP-1 and MMP-3 induction reported here are distinct from the absorption spectrum of DNA and are different from results previously reported in the literature. Importantly, they overlap with wavelengths whose intensity is predicted to increase on the earth's surface upon ozone depletion. Intensities and particular wavelengths used in our studies in vitro can be absorbed readily by fibroblasts within the skin in vivo and, thus, are relevant for risk assessment and development of protective agents.

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 x 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 x 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 x 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.

The determination of pharmacokinetic parameters of ketotifen in steady state in young children.

Ketotifen kinetics at steady state in small children compared to adults show very good absorption for a dose of 2 X daily 1 mg. Relative to adult kinetics, a higher dose in relation to body weight is justified, since a more rapid kinetic pattern was proven for the substance in the volunteer collective tested.

UV-induced reactive oxygen species in photocarcinogenesis and photoaging.

The increase in UV irradiation on earth due to the stratospheric ozone depletion represents a major environmental threat to the skin increasing its risk of photooxidative damage by UV-induced reactive oxygen species (ROS). Increased ROS load has been implicated in several pathological states including photoaging and photocarcinogenesis of the skin. Large efforts have been made to better define the involvement of distinct ROS in photocarcinogenesis and photoaging. Both pathological processes share common features; however, they reveal unique molecular characteristics which finally determine the fate of the cell and its host. As well as causing permanent genetic changes involving protooncogenes and tumor suppressor genes, ROS activate cytoplasmic signal transduction pathways that are related to growth differentiation, senescence, transformation and tissue degradation. This review focuses on the role of UV-induced ROS in the photodamage of the skin resulting in biochemical and clinical characteristics of photocarcinogenesis and photoaging. A decrease in the ROS load by efficient sunscreens and/or otherwise protective agents may represent a promising strategy to prevent or at least minimize ROS induced cutaneous pathological states.

Activation of p70 ribosomal protein S6 kinase is an essential step in the DNA damage-dependent signaling pathway responsible for the ultraviolet B-mediated increase in interstitial collagenase (MMP-1) and stromelysin-1 (MMP-3) protein levels in human dermal fibroblasts.

Ultraviolet B (UVB) irradiation has been shown to stimulate the expression of matrix-degrading metalloproteinases via generation of DNA damage and/or reactive oxygen species. Matrix-degrading metalloproteinases promote UVB-triggered detrimental long term effects like cancer formation and premature skin aging. Here, we were interested in identifying components of the signal transduction pathway that causally link UVB-mediated DNA damage and induction of matrix-degrading metalloproteinase (MMP)-1/interstitial collagenase and MMP-3/stromelysin-1 in human dermal fibroblasts in vitro. The activity of p70 ribosomal S6 kinase, a downstream target of the FK506-binding protein-12/rapamycin-associated protein kinase (FRAP) kinase (RAFT1, mTOR), was identified to be 4.8 +/- 0.8-fold, and MMP-1 and MMP-3 protein levels 2.4- and 11.5-fold increased upon UVB irradiation compared with mock-irradiated controls. The FRAP kinase inhibitor rapamycin and the DNA repair inhibitor aphidicolin significantly suppressed the UVB-mediated increase in p70 ribosomal S6 kinase activity by 50-65% and MMP-1 and MMP-3 protein levels by 34-68% and 42-88% compared with UVB-irradiated fibroblasts. By contrast, the interleukin-1beta-mediated increase in MMP-1 and MMP-3 protein levels could not be suppressed by rapamycin. Collectively, our data suggest that the FRAP-controlled p70 ribosomal S6 kinase is an essential component of a DNA damage-dependent, but not of the interleukin-1/cell membrane receptor-dependent signaling.

Increased deposition of fibulin-2 in solar elastosis and its colocalization with elastic fibres.

BACKGROUND: Fibulin-2 is a 195-kDa protein belonging to a novel family of extracellular matrix proteins that might be involved in microfibril and elastic fibre organization. OBJECTIVES: To determine the localization of fibulin-2 in relation to elastic fibres in normal skin and in solar elastosis characterized by increased elastotic material in the papillary dermis. METHODS: The expression and synthesis of fibulin-2 was investigated by means of in situ hybridization, immunohistochemistry and Western blot analysis in normal and photoaged skin. RESULTS: Immunohistochemistry and elastic tissue staining revealed that fibulin-2 deposition mainly colocalized with microfibrils and elastin fibres, with a marked staining of elastotic material in solar elastosis. Western blot analysis demonstrated that in photoaged skin fibulin-2 showed the same electrophoretic mobility as in sun-protected skin. However, in actinic elastosis the amount of fibulin-2 was significantly higher. In addition, smaller degradation products were detectable, presumably reflecting increased proteinase activity in photodamaged skin. CONCLUSIONS: This study shows that deposition of fibulin-2 and elastin is highly co-ordinated, indicating that this protein plays an important role in elastic fibre and microfibril formation in normal and actinically damaged skin.

Long-term growth arrest of PUVA-treated fibroblasts in G2/M in the absence of p16(INK4a) p21(CIP1) or p53.

Premature aging of the skin is a prominent side-effect of psoralen photoactivation, a therapy used for different skin disorders. Recently, we demonstrated that treatment of fibroblasts with 8-methoxypsoralen and ultraviolet A irradiation resulted in growth arrest with morphological and functional changes reminiscent of replicative senescence. To further elucidate the underlying molecular mechanisms, we analysed the cell-cycle phases of the growth-arrested fibroblasts. After PUVA treatment, fibroblasts arrested in G2/M, in contrast to spontaneously senesced fibroblasts arresting in a cell-cycle phase with many features similar to G1. To address the role of the cell-cycle controlling genes p16(INK4a), p21(CIP1) and p53, we analysed the expression of these genes. p16(INK4a), p21(CIP1) and p53 protein levels increased substantially with different time kinetics in growth-arrested fibroblasts. Because p16(INK4a), p21(CIP1) and p53 are involved in replicative senescence, we applied the PUVA regimen to fibroblasts deficient in either of these genes. p16(INK4a), p21(CIP1) and p53 null mutant fibroblast strains underwent growth arrest with a senescent phenotype similar to wild-type human fibroblasts. Based on these results, we propose that redundant or alternate pathways are involved in the response of dermal fibroblasts to PUVA treatment resulting in a phenocopy of replicative senescence in vitro.