The purpose of this study is to better understand the roles of the p53 tumor suppressor protein and the product of the p53-regulated gene p21 WAF1 in the response of diploid human dermal ®broblast cultures to 254 nm ultraviolet (UV) light. We report that Li ± Fraumeni syndrome (LFS) ®broblast strains heterozygous for TP53 mutation at either codon 245 or 234 exhibit markedly reduced or no expression of p21 WAF1 following UV irradiation, respectively. These strains also exhibit defective nucleotide excision repair and pronounced inhibition of RNA synthesis following UV exposure, both of which are molecular hallmarks of cells derived from patients with the UV-sensitive syndrome xeroderma pigmentosum. In sharp contrast to xeroderma pigmentosum cells, however, the repair-de®cient LFS cells show abnormal resistance, rather than hypersensitivity, to the killing eect of UV light. We further demonstrate that exposure of normal human ®broblasts to biologically relevant¯uences (415 J/m 2 ) of UV does not induce apoptotic cell death, indicating that UV resistant phenotype displayed by LFS strains is not associated with deregulated apoptosis. In normal ®broblasts, such treatment results in a moderate (*threefold) up-regulation of p53 protein, induction of the p21 WAF1 gene, and a senescence-like growth arrest. On the other hand, exposure to 520 J/m 2 UV results in a striking up-regulation of p53, inhibition of p21 WAF1 expression, and activation of an apoptotic pathway. We conclude that: (i) p21 WAF1-mediated senescence is the principal mode of cell death induced by 415 J/m 2 UV light in normal human ®broblasts; (ii) there is a threshold eect for p53-dependent apoptosis and that, in normal human cells, this threshold level is induced upon expsoure to *20 J/m 2 UV; (iii) the p53 signaling pathway is malfunctional in the TP53 heterozygous LFS strains examined; and (iv) the enhanced resistance to UV-induced cell killing displayed by these LFS strains is a consequence of diminished growth arrest, which is presumably mediated by p21 WAF1 and not abnormalities in an apoptotic pathway.

Dermal fibroblast strains cultured from affected members of a cancer-prone family with Li-Fraumeni syndrome (LFS) harbor a point mutation in one allele of the p53 tumor suppressor gene, resulting in loss of normal p53 function. In this study we have examined the ability of these p53-deficient strains to carry out the long-patch mode of excision repair, mediated by DNA polymerases delta and epsilon, after exposure to 60Co gamma radiation or far ultraviolet (UV) (chiefly 254 nm) light. Repair was monitored by incubation of the irradiated cultures in the presence of aphidicolin (apc) or 1-beta-D-arabinofuranosylcytosine (araC), each a specific inhibitor of long-patch repair, followed by measurement of drug-induced DNA strand breaks (reflecting non-ligated strand incision events) by alkaline sucrose velocity sedimentation. The LFS strains displayed deficient repair capacity in response to both gamma rays and UV light. The repair anomaly in UV-irradiated LFS cultures was manifested not only in the overall genome, but also in the transcriptionally active, preferentially repaired c-myc gene. Using autoradiography we also assessed unscheduled DNA synthesis (UDS) after UV irradiation and found this conventional measure of repair replication to be deficient in LFS strains. Moreover, both apc and araC decreased the level of UV-induced UDS by approximately 75% in normal cells, but each had only a marginal effect on LFS cells. We further demonstrated that the LFS strains are impaired in the recovery of both RNA and replicative DNA syntheses after UV treatment, two molecular anomalies of the DNA repair deficiency disorders xeroderma pigmentosum and Cockayne's syndrome. Together these results imply a critical role for wild-type p53 protein in DNA polymerase delta/epsilon-mediated excision repair, both the mechanism operating on the entire genome and that acting on expressed genes.

Osteoarthritis is a debilitating joint disease where the articular cartilage surface degrades and is unable to repair itself through natural processes. Chondrocytes reside within the cartilage matrix and maintain its structure. We conducted in vitro experiments to investigate the morphological response of cultured human chondrocytes under different pulsed electromagnetic field (PEMF) conditions. In the control experiments, cultured chondrocytes attached to the bottom of a culture dish typically displayed either a stellate or spindle morphology with extended processes. Experimental chondrocyte cultures were placed in a Helmholtz coil to which a ramp waveform was applied. Exposure to PEMFs caused the chondrocytes to retract their processes, becoming spherical in shape. This change in morphology followed a progression from stellate to spindle to spherical. These morphological changes were reflected in an average reduction of 30% in the surface contact area of the chondrocytes to the culture dish. Understanding the mechanisms by which PEMFs affect the morphology of chondrocytes will help lead to new treatments for osteoarthritis.

Studies involving disease progression in osteoarthritis (OA) have typically focused on the deterioration of native articular cartilage (AC) rather than the de novo cartilage which is frequently present. In general, there are two categories of de novo tissue observed in OA: (1) a pannus-like fibrocartilage that overlays native AC and (2) osteophytes. In this study, 30 AC samples representing a range of disease stages consistent with early to intermediate OA were examined for the occurrence of pannus-like tissue. All AC samples were examined immunohistochemically and compared with cartilage from three mature-looking osteophytes. To accomplish this, serial cartilage sections, derived from total knee arthroplasty specimens, were stained with hematoxylin and eosin and probed with antibodies raised against collagen type I, collagen type II, and aggrecan. Pannus-like tissue ranging from fibrous tissue to fibrocartilage was observed in 3 out of 30 AC samples. The appearance of this tissue was restricted to cartilage displaying signs of intermediate deterioration consistent with Outerbridge grade 2. Collagen type I, collagen type II, and aggrecan were abundant in both pannus-like tissue and osteophyte cartilage. In OA, the intrinsic repair process can yield a range of tissue types between fibrous tissue and fibrocartilage that is well integrated with the underlying, eroded AC. The absence of repair tissue from osteoarthritic samples representing the early stages of AC deterioration indicated that a relationship exists between macroscopic damage and a localized cellular repair response. Several histological and immunohistochemical similarities were also observed between the pannus-like tissue and osteophyte-derived cartilage, suggesting a common developmental process.

Ataxia telangiectasia (AT) is an autosomal recessive human disorder featuring diverse clinical abnormalities including proneness to cancer and extreme sensitivity to ionizing radiation. Although cells from AT patients exhibit faulty activation of the p53 signal transduction pathway at early times after radiation exposure, it has been proposed that high levels of DNA damage persisting in AT cells may up-regulate p53 through an ATM-independent mechanism at late times after irradiation, leading to cell death by apoptosis. In this study we demonstrate that diploid skin fibroblast strains homozygous for the AT mutation fail to up-regulate p53 protein at late times (< or = 48 h) after irradiation with 60Co gamma rays. Moreover, exposure of normal and AT fibroblasts to a dose of 8 Gy does not result in a significant increase in the fraction of apoptotic cells. Since this treatment reduces the clonogenic potential of human cells by at least two orders of magnitude, we conclude that apoptosis is not the primary mechanism of cell death induced by ionizing radiation in human normal and AT fibroblast cultures. Therefore, our results are not in accordance with the current hypothesis suggesting that increased radiosensitivity of AT cells is associated with deregulated apoptosis.