UV-induced Cell Death Research Articles

Resistance to UV-induced cell-killing in nucleophosmin/B23 over-expressed NIH 3T3 fibroblasts: enhancement of DNA repair and up-regulation of PCNA in association with nucleophosmin/B23 over-expression

Nucleophosmin/B23 was rapidly up-regulated after UV irradiation as p53, PCNA and c-Jun. UV induction of nucleophosmin/B23 was evidently increased at 3 h post-irradiation, and reached a maximum at 12 h, and remained high for at least 24 h. Over-expression of nucleophosmin/B23 made cells more resistant to UV-induced cell growth inhibition and death as compared with control vector-transfected cells through three main observations: cell growth/death percentage determination; clonogenic survival assay; and flow cytometric analysis. Moreover, nucleophosmin/B23 over-expressed cells had a greater capacity to repair UV-damaged reporter plasmid, indicating a higher nucleotide excision repair (NER) activity. Furthermore, PCNA, an essential component for DNA repair machinery, was correlated with nucleophosmin/B23 expression. Both protein level and promoter activity of PCNA were higher in nucleophosmin/B23 over-expressed cells than in control vector-transfected cells. On the other hand, treatment of cells with nucleophosmin/B23 antisense oligonucleotides decreased nucleophosmin/B23 and PCNA proteins, and potentiated the UV-induced cell killing. The effect of PCNA up-regulation may be one of the reasons that nucleophosmin/B23 over-expression made cells resistant to UV-induced growth inhibition and cell-killing.

Evidence implicating a mid-region sequence of IGFBP-3 in its specific IGF-independent actions.

Insulin-like growth factor binding protein-3 (IGFBP-3) is one of six high affinity-binding proteins that share a common function in regulating the bioavailability of the insulin-like growth factors. The six binding proteins have highly conserved C- and N-terminals that are essential to this function. Additionally, they all have specific functions on cellular homeostasis independent to the regulation of the insulin-like growth factors. It has previously been shown that insulin-like growth factor binding protein-3 can accentuate UV-induced apoptosis in a human carcinoma cell line. Using the KYSE 190 oesophageal carcinoma cell line we have demonstrated that a 15 amino acid (aa) peptide that lies within the mid-region of the protein can mimic the effect of the intact protein. This region contains the serine residues Ser(111) and Ser(113). Using two protocols, we modified these serine residues and have shown that both phosphorylation and derivatization of IGFBP-3 can negate the accentuation of UV-induced cell death. These three independent pieces of evidence support the hypothesis that the variable mid-region is responsible for the specific pro-apoptotic functions of IGFBP-3, and suggest that phosphorylation may provide a mechanism for regulation of this action.

Differential Involvement of Initiator Caspases in Apoptotic Volume Decrease and Potassium Efflux during Fas- and UV-induced Cell Death

Caspase activation and apoptotic volume decrease are fundamental features of programmed cell death; however, the relationship between these components is not well understood. Here we provide biochemical and genetic evidence for the differential involvement of initiator caspases in the apoptotic volume decrease during both intrinsic and extrinsic activation of apoptosis. Apoptosis induction in Jurkat T lymphocytes by Fas receptor engagement (intrinsic) or ultraviolet (UV)-C radiation (extrinsic) triggered the loss of cell volume, which was restricted to cells with diminished intracellular K(+) ions. These characteristics kinetically coincided with the proteolytic processing and activation of both initiator and effector caspases. Although the polycaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone completely inhibited the Fas-mediated apoptotic volume decrease and K(+) efflux, it was much less effective in preventing these processes during UV-induced cell death under conditions whereby caspase activities and DNA degradation were blocked. To define the roles of specific initiator caspases, we utilized Jurkat cells genetically deficient in caspase-8 or stably transfected with a dominant-negative mutant of caspase-9. The results show that the activation of caspase-8, but not caspase-9, is necessary for Fas-induced apoptosis. Conversely, caspase-9, but not caspase-8, is important for UV-mediated shrunken morphology and apoptosis progression. Together, these findings indicate that cell shrinkage and K(+) efflux during apoptosis are tightly coupled, but are differentially regulated by either caspase-8 or caspase-9 depending on specific pathways of cell death.

Heat shock proteins in the photobiology of human skin

All organisms respond to sudden environmental changes with the increased transcription of genes belonging to the family of heat shock proteins (hsps). Hsp-inducing stress factors include elevated temperatures, alcohol, heavy metals, oxidants, and agents leading to protein denaturation. The induction of heat shock proteins is followed by a transient state of increased resistance to further stress and the heat shock response is generally thought to represent an evolutionary conserved adaptive mechanism to cope with hostile environmental conditions. Since the skin as a barrier organ has to cope with the potentially harmful consequences of exposure to ultraviolet radiation (UV), it appears reasonable to question whether hsps constitute a natural defence mechanism against UV. Hsps have been detected in resting as well as in stressed epidermal and dermal cells and overexpression of hsps is associated with increased resistance to UV-induced cell death. Furthermore, UV itself is able to induce the expression of specific hsps. Thus, hsps might provide an adaptive cellular response to increasing UV and enhancing the expression of hsps might turn out as a new way to deal with the immediate and long-term consequences of UV exposure. Prerequisite for the utilization of this concept is the development of non-toxic heat shock inducers and their evaluation for clinical efficacy and safety.

Implication of the Small GTPase Rac1 in the Apoptosis Induced by UV in Rat-2 Fibroblasts

Exposure of mammalian cells to ultraviolet (UV) light elicits a cellular response and also lead to apoptotic cell death. However, the role of Rac, a member of Rho family GTPases, in the UV-induced apoptosis has never been examined. In UV-irradiated Rat-2 fibroblasts, nuclear fragmentation began to be observed within 2 h and the total viability of Rat-2 cells were only about 15% at 6 h following by UV irradiation, whereas the total viability in Rat2-RacN17 cells stably expressing RacN17, a dominant negative Rac1 mutant, was almost close to 67%. Pretreatment with SB203580, a specific inhibitor of p38 kinase, likewise attenuated UV-induced cell death, but PD98059, a MEK inhibitor, did not. Thus, Rac1 and p38 kinase appear to be components in the apoptotic signaling pathway induced by UV irradiation in Rat-2 fibroblasts. In addition, our results show that p38 kinase stimulation by UV is dramatically inhibited by RacN17, suggesting that p38 kinase is situated downstream of Rac1 in the UV signaling to apoptosis.

Direct inhibition of caspase 3 is dispensable for the anti-apoptotic activity of XIAP.

XIAP is a mammalian inhibitor of apoptosis protein (IAP). To determine residues within the second baculoviral IAP repeat (BIR2) required for inhibition of caspase 3, we screened a library of BIR2 mutants for loss of the ability to inhibit caspase 3 toxicity in the yeast Schizosaccharomyces pombe. Four of the mutations, not predicted to affect the structure of the BIR fold, clustered together on the N-terminal region that flanks BIR2, suggesting that this is a site of interaction with caspase 3. Introduction of these mutations into full-length XIAP reduced caspase 3 inhibitory activity up to 500-fold, but did not affect its ability to inhibit caspase 9 or interact with the IAP antagonist DIABLO. Furthermore, these mutants retained full ability to inhibit apoptosis in transfected cells, demonstrating that although XIAP is able to inhibit caspase 3, this activity is dispensable for inhibition of apoptosis by XIAP in vivo.

Inhibition of experimental vasospasm by pretreatment with ultraviolet light irradiation in a rat femoral artery model.

Chronic cerebral vasospasm is resistant to conventional treatments despite recent advances in treatment modalities. We studied the preventive effect of ultraviolet (UV) irradiation on development of vasospasm and its mechanism in a rat femoral artery model. The rat femoral artery model for vasospasm was used in this investigation (n = 108). The femoral arteries were divided into four groups: empty and no irradiation (control), UV irradiation (UV group), blood placement (VS group), and blood placement after UV irradiation (VS + UV group). Luminal area was measured, and smooth muscle cell counts in the medial layer of the vessel wall were obtained. An immunohistochemical study was performed with cross sections of fixed femoral arteries at 12 hours and 1, 3, 5, 7, and 49 days after blood placement. The rings of femoral arteries on Day 7 were subjected to pharmacological study. Pretreatment with UV irradiation (VS + UV group) resulted not only in significant inhibition of chronic vasospasm but also in a significant decrease in smooth muscle cells compared with the VS group on Days 5 and 7. The UV-treated arteries (UV and VS + UV groups) exhibited a significant number of Bax- and Bcl-2-positive cells on Days 5 and 7, but few CPP-32 positive cells were observed at the same time points. In the pharmacological study, contractile response to KCI or phenylephrine was reduced significantly in the UV-treated arteries. These results imply that UV irradiation prevents chronic vasospasm and suggest that UV-induced cell death plays an important role in the preventive effect without causing complications during the chronic period.

Bax gene disruption alters the epidermal response to ultraviolet irradiation and in vivo induced skin carcinogenesis.

Bcl-2 family member proteins are differentially expressed in skin and in non-melanoma skin cancer (NMSC). To elucidate the contribution of bcl-2 and bax proteins to epidermal differentiation and skin carcinogenesis, we investigated keratinocyte proliferation, differentiation and tumourigenesis in bcl-2(-/-) and bax(-/-) mice. The rate and pattern of proliferation and spontaneous cell death in the bcl-2(-/-) and bax(-/-) mice were not different from control mice. The epidermis of bcl-2(-/-) and bax(-/-) expressed sightly higher levels of cytokeratin 1 and loricrin compared to control littermates. The apoptotic response to ultraviolet-induced genotoxic stress was assessed by quantitating TUNEL positive cells. Bax(-/-) keratinocytes showed a significant resistance to UV-induced cell death compared to control mice. The life-span of bcl-2(-/-) mice precluded an assessment of bcl-2 gene disruption on in vivo tumourigenesis. A significant increase in tumour incidence was observed in bax(-/-) mice compared to control mice in two-step chemical carcinogenesis studies. These findings suggest that bcl-2 and bax gene products may be important determinants of normal keratinocyte differentiation and response to genotoxic stress in vivo, and indicate that bax may provide a tumour suppressor effect during skin carcinogenesis.

Disruption of the Cockayne syndrome B gene impairs spontaneous tumorigenesis in cancer-predisposed Ink4a/ARF knockout mice.

Cells isolated from individuals with Cockayne syndrome (CS) have a defect in transcription-coupled DNA repair, which rapidly corrects certain DNA lesions located on the transcribed strand of active genes. Despite this DNA repair defect, individuals with CS group A (CSA) or group B (CSB) do not exhibit an increased spontaneous or UV-induced cancer rate. In order to investigate the effect of CSB deficiency on spontaneous carcinogenesis, we crossed CSB(-/-) mice with cancer-prone mice lacking the p16(Ink4a)/p19(ARF) tumor suppressor locus. CSB(-/-) mice are sensitive to UV-induced skin cancer but show no increased rate of spontaneous cancer. CSB(-/-) Ink4a/ARF(-/-) mice developed 60% fewer tumors than Ink4a/ARF(-/-) animals and demonstrated a longer tumor-free latency time (260 versus 150 days). Moreover, CSB(-/-) Ink4a/ARF(-/-) mouse embryo fibroblasts (MEFs) exhibited a lower colony formation rate after low-density seeding, a lower rate of H-Ras-induced transformation, slower proliferation, and a lower mRNA synthesis rate than Ink4a/ARF(-/-) MEFs. CSB(-/-) Ink4a/ARF(-/-) MEFs were also more sensitive to UV-induced p53 induction and UV-induced apoptosis than were Ink4a/ARF(-/-) MEFs. In order to investigate whether the apparent antineoplastic effect of CSB gene disruption was caused by sensitization to genotoxin-induced (p53-mediated) apoptosis or by p53-independent sequelae, we also generated p53(-/-) and CSB(-/-) p53(-/-) MEFs. The CSB(-/-) p53(-/-) MEFs demonstrated lower colony formation efficiency, a lower proliferation rate, a lower mRNA synthesis rate, and a higher rate of UV-induced cell death than p53(-/-) MEFs. Collectively, these results indicate that the antineoplastic effect of CSB gene disruption is at least partially p53 independent; it may result from impaired transcription or from apoptosis secondary to environmental or endogenous DNA damage.

Reduction of UV-induced Cell Death in the Human Senescent Fibroblasts

Reduction of UV-induced Cell Death in the Human Senescent Fibroblasts

Increase in ultraviolet sensitivity by overexpression of calpastatin in ultraviolet-resistant UVr-1 cells derived from ultraviolet-sensitive human RSa cells.

Human RSa cells are highly sensitive to apoptotic-like cell death by ultraviolet irradiation (UV) while UVr-1 cells are their variant with an increased resistance to UV. Three days after UV at 10 J/m2, the viability of RSa cells was approximately 17% while that of UVr-1 cells was 65%. This different survival might reflect apoptotic cell death since apoptosis-specific DNA ladder was more clearly observed in RSa cells than in UVr-1 cells after UV. Addition of ALLN/calpain inhibitor I to the culture medium after UV resulted in similar survival (14 - 18%) between RSa and UVr-1 cells. Immunoblot analysis showed down-regulation of protein kinase CTheta, Src, Bax and mu-calpain after UV was more prominent in UVr-1 than in RSa cells. Activated mu-calpain appeared within 1 h post-UV only in UVr-1 cells. The expression of calpastatin, a specific endogenous inhibitor of calpain, was higher in RSa than in UVr-1 cells. To further examine the role of calpain in UV-induced cell death, cDNA of human calpastatin was transfected into UVr-1 cells. The results showed that overexpression of calpastatin suppressed down-regulation of Src, mu-calpain and Bax. Concomitantly, colony survival after UV was reduced in calpastatin-transfected cells as compared to vector control cells. Our results suggest that activation of calpain might account for, at least in part, the lower susceptibility to UV-induced cell death in UVr-1 cells.

Role of TRAF2/GCK in melanoma sensitivity to UV-induced apoptosis.

Radiation resistance is a hallmark of human melanoma, and yet mechanisms underlying this resistance are not well understood. We recently established the role of ATF2 in this process, suggesting that stress kinases, which contribute to regulation of ATF2 stability and activity, play an important role in the acquisition of such resistance. Here we demonstrate that changes in the expression and respective activities of TRAF2/GCK occur during melanoma development and regulate its sensitivity to UV-induced apoptosis. Comparing early- and late-stage melanoma cells revealed low expression of TRAF2 and GCK in early-stage melanoma, which coincided with poor resistance to UV-induced, TNF-mediated apoptosis; forced expression of GCK alone or in combination with TRAF2 efficiently increased JNK and NF-kappaB activities, which coincided with increased protection against apoptosis. Conversely, forced expression of the dominant negative form of TRAF2 or GCK in late-stage melanoma cells reduced NF-kappaB activity and decreased Fas expression, resulting in a lower degree of UV-induced, Fas-mediated cell death. Our results illustrate a mechanism in which protection from, or promotion of, UV-induced melanoma cell death depends on the nature of the apoptotic cascade (TNF or Fas) and on the availability of TRAF2/GCK, whose expression increases during melanoma progression. Oncogene (2000) 19, 933 - 942.

C-Jun regulates cell cycle progression and apoptosis by distinct mechanisms.

c-Jun is a component of the transcription factor AP-1, which is activated by a wide variety of extracellular stimuli. The regulation of c-Jun is complex and involves both increases in the levels of c-Jun protein as well as phosphorylation of specific serines (63 and 73) by Jun N-terminal kinase (JNK). We have used fibroblasts derived from c-Jun null embryos to define the role of c-Jun in two separate processes: cell growth and apoptosis. We show that in fibroblasts, c-Jun is required for progression through the G1 phase of the cell cycle; c-Jun-mediated G1 progression occurs by a mechanism that involves direct transcriptional control of the cyclin D1 gene, establishing a molecular link between growth factor signaling and cell cycle regulators. In addition, c-Jun protects cells from UV-induced cell death and cooperates with NF-kappaB to prevent apoptosis induced by tumor necrosis factor alpha (TNFalpha). c-Jun mediated G1 progression is independent of phosphorylation of serines 63/73; however, protection from apoptosis in response to UV, a potent inducer of JNK/SAP kinase activity, requires serines 63/73. The results reveal critical roles for c-Jun in two different cellular processes and show that different extracellular stimuli can target c-Jun by distinct biochemical mechanisms.

Alterations in cell death and cell cycle progression in the UV-irradiated epidermis of bcl-2-deficient mice.

The effect of bcl-2 gene ablation on epidermal cell death induced by UV-B irradiation was investigated in mice. Exposure of depilated back skin of bcl-2-/- mice to 0.5 J/cm2 UV-B caused a prolonged increase in the number of epidermal cells showing nuclear DNA fragmentation compared to wild-type littermates. Consistently, skin explants from bcl-2-deficient mice exhibited a higher number of sunburn cells per cm epidermis (16.6+/-2.1 vs 7.0+/-1.5) following exposure to 0.1 J/cm2 UV-B in vitro. Furthermore, UV irradiation failed to increase pre-melanosomes in skin explants from mutant animals, and primary menalocyte cultures derived from bcl-2 null mutants were highly susceptible to UV-induced cell death compared to cultures from wild-type littermates. An accelerated reappearance of proliferating cells, showing nuclear immunoreactivity for Ki-67 and c-Fos, was observed in the UV-irradiated epidermis of bcl-2-deficient mice. Taken together, these findings suggest that effects of UV radiation on epidermal cell death and cell cycle progression are influenced by survival-promoting Bcl-2.

The phototoxic effects of UVB (311 nm) and psoralen in PUVB bath therapy

Exposure to ultraviolet (UV) radiation and photochemotherapy induces apoptotic cell death in epidermal cells. In this study annexin V binding and propidium iodide (PI) uptake have been measured by flow cytometry to evaluate UV-induced cell death in the human squamous cell carcinoma-derived cell line A 431. Physiological and therapeutical relevant doses of UVA, UVA1, UVB, narrow-band UVB (311 nm) and photochemotherapy using 100 ng/ml of 8-methoxypsoralen (8-MOP) with UVA or UVA1 (PUVA or PUVA1) have been applied. Doses ranged from 8 to 96 J/cm2 for UVA1 and UVA, from 8 to 128 mJ/cm2 for UVB, from 256 to 4096 mJ/cm2 for narrow-band UVB (311 nm) and from 1 to 16 J/cm2 for photochemotherapy. Results show that the amount of annexin V binding, a measure of early apoptosis, as well as PI uptake, a parameter of ultimate cell death, are strictly correlated with the applied UV dose. Peak values of annexin V-positive cells are noted 12 h after UV exposure in all protocols and are followed by an increase of PI-uptaking cells with peak values at 24 h after UVA and UVA1, and 48 h after PUVA, PUVA1, UVB and narrow-band UVB. To compare the effect of different wavelengths and light sources, dose equivalents are calculated based on the induction of 50% cell death (as determined by PI uptake). The equivalents are 96 J/ cm2 for UVA and UVA1, 16 J/cm2 for PUVA and PUVA1, 256 mJ/cm2 for UVB and 2048 mJ/cm2 for narrow-band UVB. Our results establish annexin V/PI double staining as an appropriate method for the quantification of UV-induced cell death. Moreover, they provide a basis for further investigations concerning mechanisms and modifications of UV-induced apoptosis.

P53-Regulated Apoptosis Is Differentiation Dependent in Ultraviolet B-Irradiated Mouse Keratinocytes

Previous studies from our laboratory, using p53 transgenic mice, have suggested that ultraviolet (UV) light-induced keratinocyte apoptosis in the skin is not affected by overexpression of mutant p53 protein. To further elucidate a possible role for p53 in UV-induced keratinocyte cell death, we now examine apoptosis in skin and isolated keratinocytes from p53 null (-/-) mice and assess the influence of cell differentiation on this process. In vivo, using this knockout model, epidermal keratinocytes in p53-/- mice exhibited only a 5.2-fold increase in apoptosis after 2000 J/m2 UVB irradiation compared with a 26.3-fold increase in normal control animals. If this p53-dependent apoptosis is important in elimination of precancerous, UV-damaged keratinocytes, then it should be active in the undifferentiated cells of the epidermal basal layer. To test this hypothesis, we examined the effect of differentiation on UV-induced apoptosis in primary cultures of murine and human keratinocytes. Apoptosis was p53-independent in undifferentiated murine keratinocytes, which exhibited relative resistance to UVB-induced killing with only a 1.5-fold increase in apoptosis in p53+/+ cells and a 1.4-fold increase in p53-/- cells. Differentiated keratinocytes, in contrast, showed a 9.4-fold UVB induction of apoptosis in p53+/+ cells, almost three times the induction observed in p53-/- cells. This UV-induced difference in apoptosis was observed when keratinocytes were cultured on type IV collagen substrate, but not on plastic alone. Western blotting of UV-irradiated, differentiated keratinocytes did not support a role for either Bax or Bcl-2 in this process. In support of these findings in mice, cell death in human cultured keratinocytes also occurred in a differentiation-associated fashion. We conclude that p53-induced apoptosis eliminates damaged keratinocytes in the differentiated cell compartment, but this mechanism is not active in the basal, undifferentiated cells and is therefore of questionable significance in protection against skin cancer induction.

Flow cytometric quantification of UY-induced cell death in a human squamous cell carcinoma-derived cell line: dose and kinetic studies

Exposure to ultraviolet (UV) radiation and photochemotherapy induces apoptotic cell death in epidermal cells. In this study annexin V binding and propidium iodide (PI) uptake have been measured by flow cytometry to evaluate UV-induced cell death in the human squamous cell carcinoma-derived cell line A 431. Physiological and therapeutical relevant doses of UVA, UVA1, UVB, narrow-band UVB (311 nm) and photochemotherapy using 100 ng/ml of 8-methoxypsoralen (8-MOP) with UVA or UVA1 (PUVA or PUVA1) have been applied. Doses ranged from 8 to 96 J/cm 2 for UVA1 and UVA, from 8 to 128 mJ/cm 2 for UVB, from 256 to 4096 mJ/cm 2 for narrow-band UVB (311 nm) and from 1 to 16 J/cm 2 for photochemotherapy. Results show that the amount of annexin V binding, a measure of early apoptosis, as well as PI uptake, a parameter of ultimate cell death, are strictly correlated with the applied UV dose. Peak values of annexin V-positive cells are noted 12 h after UV exposure in all protocols and are followed by an increase of PI-uptaking cells with peak values at 24 h after UVA and UVA1, and 48 h after PUVA, PUVA1, UVB and narrow-band UVB. To compare the effect of different wavelengths and light sources, dose equivalents are calculated based on the induction of 50% cell death (as determined by PI uptake). The equivalents are 96 J/ cm 2 for UVA and UVA1, 16 J/cm 2 for PUVA and PUVA1, 256 mJ/cm 2 for UVB and 2048 mJ/cm 2 for narrow-band UVB. Our results establish annexin V/PI double staining as an appropriate method for the quantification of UV-induced cell death. Moreover, they provide a basis for further investigations concerning mechanisms and modifications of UV-induced apoptosis.

Location and Function of Erythropoietin (Epo) and its Receptor (Epo-R) in the Developing Human Brain • 1876

We have previously identified the presence of Epo and its receptor in the human fetal CNS, and reported that pharmacologic doses of rEpo decrease hypoxia-induced apoptosis in human neuronal precursor cells (NT2). We now describe specific changes in the cellular distribution of Epo and Epo-R during neurodevelopment. We also report that physiologic concentrations of rEpo protect NT2 cells from UV-induced apoptosis. Two antibodies against human Epo-R, and Epo were used to identify immunoreactive cells in brains at 5, 10, 20, 24, 28, 32 weeks post conception, and in adult brains. The specificity of antibody reactions were checked by Western analysis. Controls for immunohistochemistry included specific and non-specific blocking peptides, absence of primary antibody, and substitution of pre-immune serum for specific antibody. At 5 weeks gestation, anti-Epo and Epo-R antibodies were both immunoreactive in the ventricular zone, while at 10 weeks only Epo staining was prominent in the ventricular zone, with Epo-R staining primarily the subventricular zone. Both antibodies stained the cortical plate. As development progressed, Epo reactivity became most prominent in neurons, although a subpopulation of astrocytes were also immunoreactive. This was in contrast to Epo-R staining, which was most prominent in astrocytes, although weak immunoreactivity was also present on neurons. To test the hypothesis that Epo is neuroprotective at physiologic doses, we pretreated NT2 cells for 16 hrs with either 50mU/mL rEpo, 50mU/mL rEpo+neutralizing antibody, or PBS(n=8). Cells were exposed to 11 sec UV irradiation (vs no UV control). After 16 hrs, cells were stained with trypan blue and counted, and nuclear matrix protein (NMP) released into the media was measured by ELISA. Cells exposed to 50 mU/mL Epo were rescued from UV-induced apoptotic cell death, as evaluated by both cell counts and decreased NMP (p<0.05). We have previously documented Epo concentration in neonatal CSF as high as 68 mU/mL, thus 50 mU/mL is within the physiologic range. We conclude that Epo and its receptor are present within the developing human brain, that the pattern of distribution of these proteins change with development, and that inhibiting apoptotic cell death may be one function of this cytokine-ligand pair.

Overexpression of the small heat shock protein, hsp27, confers resistance to hyperthermia, but not to oxidative stress and UV-induced cell death, in a stably transfected squamous cell carcinoma cell line

The 27 kD heat shock protein (hsp27) is expressed in human keratinocytes in association with differentiation in vitro and in situ. This study was conducted to investigate whether the expression of hsp27 in keratinocytes is associated with increased resistance to the deleterious effects of heat and UV radiation. A transfection vector carrying the human gene for hsp27, under the control of hsp27 as well as the SV40 promoter (pSG2711, M. Jäättelä et al., EMBO J. 11 (1992) 3507–3512), was introduced together with a neomycin-resistance gene into the squamous cell carcinoma cell line A431. Cells were exposed to either UVA, UVB, heat (45 °C, 4 h) or hydrogen peroxide (0.025-0.5 mM) and the percentage of surviving cells was determined. Overexpression of hsp27 induced increased resistance to hyperthermia, but not to hydrogen peroxide-mediated oxidative injury. When cells were exposed to increasing amounts of UVA (5–80 J cm −2) and UVB (4–64 mJ cm −2), the percentage of surviving cells was identical for clones overexpressing hsp27 and control clones. From these data, we conclude that hsp27 is a mediator of thermotolerance, but does not protect keratinocytes from UV-induced cell death.

Induction of apoptotic cell death in hen granulosa cells by ceramide.

Recent studies have demonstrated that ovarian follicle atresia occurs extensively before follicle selection into the avian preovulatory hierarchy, and that this process is mediated via granulosa cell apoptosis. Subsequent to follicle selection, granulosa cells are inherently resistant to apoptosis, and such resistance is correlated with increased expression of death suppressor genes such as bcl-xlong. In the present studies we used this avian ovary model system to 1) identify cellular characteristics and mechanisms related to apoptotic cell death of granulosa cells in vitro, and 2) further characterize functional differences between apoptosis-susceptible (4- to 8-mm follicle) and apoptosis-resistant (preovulatory follicle) granulosa cells. Treatment of granulosa cells from the largest preovulatory follicle with N-octanoylsphingosine (C8-ceramide) results in pronounced oligonucleosome formation, a hallmark of apoptosis. That this is indicative of programmed cell death is supported by an increased incidence of pyknotic nuclei and apoptotic bodies in C8-ceramide-treated samples compared to that in control cultured cells. Tumor necrosis factor-alpha, a stimulator of ceramide production, actively promotes oligonucleosome formation in apoptosis-susceptible, but not in apoptosis-resistant, granulosa cells. Induction of apoptosis is also observed after exposure of apoptosis-resistant granulosa cells to sphingomyelinase treatment and UV irradiation, which are known to stimulate endogenous ceramide production, and to the anticancer drug, daunorubicin, which initiates de novo ceramide biosynthesis via activation of ceramide synthase. Although treatment of granulosa cells with fumonisin B1, a specific ceramide synthase inhibitor, blocks daunorubicin-stimulated oligonucleosome formation, UV-induced cell death is unaffected. Taken together, these results demonstrate that pharmacological factors known to mimic the actions of ceramide or stimulate ceramide production can induce oligonucleosome formation and programmed cell death in granulosa cells. More importantly, however, the ability of a physiologically relevant initiator of ceramide biosynthesis, tumor necrosis factor-alpha, to promote cell death is evident only in apoptosis-susceptible granulosa cells collected from atresia-prone prehierarchal follicles. These data provide support for ceramide as an important intracellular signaling mechanism, mediating granulosa cell apoptosis and follicle atresia.