Mark S. Eller

Mechanisms of Ultraviolet Light‐Induced Pigmentation

Work in the past 8 years, particularly in the past 1-2 years, has greatly expanded our understanding of the mechanisms by which ultraviolet irradiation stimulates melanogenesis in the skin. A direct effect of UV photons on DNA results in up-regulation of the gene for tyrosinase, the rate-limiting enzyme in melanin synthesis, as well as an increase in cell surface expression of receptors for at least one of the several known keratinocyte-derived melanogenic factors, MSH. Direct effects of UV on melanocyte membranes, releasing DAG and arachidonic acid, may also play a role in the tanning response. Diacylglycerol may activate PKC-beta, which in turn phosphorylates and activates tyrosinase protein; the pathways by which products of other inflammatory mediator cascades may act on melanogenesis are unknown. The tanning response also relies heavily on UV-stimulated increased production and release of numerous keratinocyte-derived factors including bFGF, NGF, endothelin-1 and the POMC-derived peptides MSH, ACTH, beta-LPH and beta-endorphin. These factors variably induce melanocyte mitosis, increase melanogenesis, enhance dendricity and prevent apoptotic cell death following the UV injury. Thus, events within the epidermal melanin unit conspire to maintain or increase melanocyte number, increase melanin pigment throughout the epidermis. Overall, ultraviolet-induced melanogenesis may be one part of a eukaryotic SOS response to damaging ultraviolet irradiation that has evolved over time to provide a protective tan in skin at risk of further injury from sun exposure. These recent insights into the mechanisms underlying ultraviolet-induced melanogenesis offer the opportunity for novel therapeutic approaches to minimizing acute and chronic photodamage in human skin.

Mechanisms and implications of the age-associated decrease in DNA repair capacity

Skin cancer incidence is clearly linked to UV irradiation and increases exponentially with age. We studied the rate of removal of thymine dimers and (6–4) photoproducts in UV‐irradiated human dermal fibroblasts derived from donors of different ages. There was a significant decrease with aging in the repair rates of both thymine dimers and (6–4) photoproducts (P< 0.001). In addition, there was an age‐associated decrease in the protein levels of ERCC3, PCNA, RPA, XPA, and p53 that participate in nucleotide excision repair. Moreover, the mRNA levels of XPA, ERCC3, and PCNA were significantly reduced with aging, suggesting that these decreases are often regulated at the mRNA level. Furthermore, with age induction of p53 after UV irradiation was significantly reduced. Taken together, our data suggest that the age‐associated decrease in the repair of UV‐induced DNA damage results at least in part from decreased levels of proteins that participate in the repair process.—Goukassian, D., Gad, F., Yaar, M., Eller, M. S., Nehal, U. S., Gilchrest, B. A. Mechanisms and implications of the age‐associated decrease in DNA repair capacity. FASEB J. 14, 1325–1334 (2000)

Evidence that exposure of the telomere 3′ overhang sequence induces senescence

Normal human cells cease proliferation after a finite number of population doublings, a phenomenon termed replicative senescence. This process, first convincingly described by Hayflick and Moorhead [Hayflick, L. & Moorhead, P. S. (1961) Exp. Cell Res. 25, 595–621] for cultured human fibroblasts 40 years ago, is suggested to be a fundamental defense against cancer. Several events have been demonstrated to induce the senescent phenotype including telomere shortening, DNA damage, oxidative stress, and oncogenic stimulation. The molecular mechanisms underlying senescence are poorly understood. Here we report that a 1-week exposure to oligonucleotide homologous to the telomere 3′-overhang sequence TTAGGG (T-oligo) similarly specifically induces a senescent phenotype in cultured human fibroblasts, mimicking serial passage or ectopic expression of a dominant negative form of the telomeric repeat binding factor, TRF2DN. We propose that exposure of the 3′ overhang due to telomere loop disruption may occur with critical telomere shortening or extensive acute DNA damage and that the exposed TTAGGG tandem repeat sequence then triggers DNA-damage responses. We further demonstrate that these responses can be induced by treatment with oligonucleotides homologous to the overhang in the absence of telomere disruption, a phenomenon of potential therapeutic importance.

The trk family of receptors mediates nerve growth factor and neurotrophin-3 effects in melanocytes.

We have recently shown that (a) human melanocytes express the p75 nerve growth factor (NGF) receptor in vitro; (b) that melanocyte dendricity and migration, among other behaviors, are regulated at least in part by NGF; and (c) that cultured human epidermal keratinocytes produce NGF. We now report that melanocyte stimulation with phorbol 12-tetra decanoate 13-acetate (TPA), previously reported to induce p75 NGF receptor, also induces trk in melanocytes, and TPA effect is further potentiated by the presence of keratinocytes in culture. Moreover, trk in melanocytes becomes phosphorylated within minutes after NGF stimulation. As well, cultures of dermal fibroblasts express neurotrophin-3 (NT-3) mRNA; NT-3 mRNA levels in cultured fibroblasts are modulated by mitogenic stimulation, UV irradiation, and exposure to melanocyte-conditioned medium. Moreover, melanocytes constitutively express low levels of trk-C, and its expression is downregulated after TPA stimulation. NT-3 supplementation to cultured melanocytes maintained in Medium 199 alone prevents cell death. These combined data suggest that melanocyte behavior in human skin may be influenced by neurotrophic factors, possibly of keratinocyte and fibroblast origin, which act through high affinity receptors.

Pigment content of cultured human melanocytes does not correlate with tyrosinase message level

Summary Tyrosinase is considered to be the rate‐limiting enzyme for the biosynthesis of melanin in epidermal melanocytes. and thus tyrosinase activity is thought to be a major regulatory step in melanogenesis. To determine whether the rate of pigment production was controlled at the level of tyrosinase gene expression. we developed a culture system capable of generating large populations of pure human melanocytes and then measured both melanin content as determined spectrophotometrically by absorption at 475 nm and mRNA levels as detected by hybridization with cloned cDNA level 34, encoding human tyrosinase. We examined the relationship between pigment content and tyrosinase mRNA levels among human melanoma and melanocyte lines with very different levels of basal pigmentation: between two clones of a single human melanoma line, one pigmented and one amelanotic, and sequentially in melanocytes before and after simulation with isoburylmethylxanthine to increase melanin content per cell. Using Northern blot analysis and in‐situ hybridization we found no correlation between tyrosinase message levels and melanin content, suggesting that post‐transcriptional regulation of tyrosinase and/or other events determine the rate of pigment synthesis in human melanocytes.

Telomere-based DNA damage responses: a new approach to melanoma

Melanoma is the most fatal skin cancer, often highly resistant to chemotherapy. Here we show that treatment with an 11‐base DNA oligonucleotide homologous to the telomere 3′ overhang sequence (T‐oligo) induces apoptosis of several established human melanoma cell lines, including the aggressive MM‐AN line, whereas normal human melanocytes exposed to the same or higher T‐oligo concentrations show only transient cell cycle arrest, implying that malignant cells are more sensitive to T‐oligo effects. When MM‐AN cells were briefly exposed to T‐oligo in culture and injected into the flank or tail vein of SCID mice, eventual tumor volume and number of metastases were reduced 85‐95% compared with control mice. Similarly, T‐oligos administered intralesionally or sys‐temically selectively inhibited the growth of previously established MM‐AN tumor nodules in the flank and peritoneal cavity by 85 to 90% without detectable toxicity. We previously showed that T‐oligos act through ATM, p95/Nbs1, E2F1, p16INK4A, p53, and the p53 homologue p73 to modulate downstream effectors and now additionally demonstrate striking down‐regulation of the inhibitor of apoptosis protein livin/ML‐IAP. We suggest that T‐oligo mimics a physiologic DNA damage signal that is frequently masked in malignant cells and thereby activates innate cancer prevention responses. T‐oligos may provide a novel therapeutic approach to melanoma.—Puri, N., Eller, M. S., Byers, H. R., Dykstra, S., Kubera, J., Gilchrest, B. A. Te‐lomere‐based DNA damage responses: a new approach to melanoma.

A role for WRN in telomere-based DNA damage responses.

Telomeres cap the ends of eukaryotic chromosomes and prevent them from being recognized as DNA breaks. We have shown that certain DNA damage responses induced during senescence and, at times of telomere uncapping, also can be induced by treatment of cells with small DNA oligonucleotides homologous to the telomere 3′ single-strand overhang (T-oligos), implicating this overhang in generation of these telomere-based damage responses. Here, we show that T-oligo-treated fibroblasts contain γH2AX foci and that these foci colocalize with telomeres. T-oligos with nuclease-resistant 3′ ends are inactive, suggesting that a nuclease initiates T-oligo responses. We therefore examined WRN, a 3′ → 5′ exonuclease and helicase mutated in Werner syndrome, a disorder characterized by aberrant telomere maintenance, premature aging, chromosomal rearrangements, and predisposition to malignancy. Normal fibroblasts and U20S osteosarcoma cells rendered deficient in WRN showed reduced phosphorylation of p53 and histone H2AX in response to T-oligo treatment. Together, these data demonstrate a role for WRN in processing of telomeric DNA and subsequent activation of DNA damage responses. The T-oligo model helps define the role of WRN in telomere maintenance and initiation of DNA damage responses after telomere disruption.

Telomere 3′ overhang-specific DNA oligonucleotides induce autophagy in malignant glioma cells

Telomere 3′ overhang‐specific DNA oligonucleotides (T‐oligos) induce cell death in cancer cells, presumably by mimicking telomere loop disruption. Therefore, T‐oligos are considered an exciting new therapeutic strategy. The purpose of this study was to elucidate how T‐oligos exert antitumor effects on human malignant glioma cells in vitro and in vivo. We demonstrated that T‐oligos inhibited the proliferation of malignant glioma cells through induction of non‐apoptotic cell death and mitochondria hyperpolarization, whereas normal astrocytes were resistant to T‐oligos. Tumor cells treated with T‐oligos developed features compatible with autophagy, with development of autophagic vacuoles and conversion of an autoph‐agy‐related protein, microtubule‐associated protein 1 light chain 3 from type I (cytoplasmic form) to type II (membrane form of autophagic vacuoles). A reversephase protein microarray analysis and Western blotting revealed that treatment with T‐oligos inhibited the mammalian target of the rapamycin (mTOR) and the signal transducer and activator of transcription 3 (STAT3). Moreover, pretreatment with T‐oligos significantly prolonged the survival time of mice inoculated intracranially with malignant glioma cells compared with that of untreated mice and those treated with control oligonucleotides (P= 0.0065 and P= 0.043, respectively). These results indicate that T‐oligos stimulate the induction of nonapoptotic autophagic also known as type II programmed cell death and are thus promising in the treatment of malignant glioma.—Aoki, H., Iwado, E., Eller, M. S., Kondo, Y., Fujiwara, K., Li, G.‐Z., Hess, K. R., Siwak, D. R., Sawaya, R., Mills, G. B., Gilchrest, B. A., Kondo, S. Telomere 3′ overhang‐specific DNA oligonucleotides induce autophagy in malignant glioma cells. FASEB J. 21, 2918–2930 (2007)

Induction of a p95/Nbs1-mediated S phase checkpoint by telomere 3′ overhang specific DNA

Telomere shortening induces a nonproliferative senescent phenotype, believed to reduce cancer risk, and telomeres are involved in a poorly understood manner in responses to DNA damage. Although telomere disruption induces p53 and triggers apoptosis or cell cycle arrest, the features of the disrupted telomere that trigger this response and the precise mechanism involved are poorly understood. Using human cells, we show that DNA oligonucleotides homologous to the telomere 3′ overhang sequence specifically induce and activate p53 and activate an S phase checkpoint by modifying the Nijmegen breakage syndrome protein, known to mediate the S phase checkpoint after DNA damage. These responses are mediated, at least in part, by the ATM kinase and are not attributable to disruption of cellular telomeres. Based on these and earlier data, we propose that these oligonucleotides mimic a physiological signal, exposure of the telomere 3′ overhang due to opening of the normal telomere loop structure, and hence evoke these protective anti‐proliferative responses in the absence of DNA damage or telomere disruption. Eller, M. S., Li, G.‐Z., Firoozabadi, R., Puri, N., Gilchrest, B. A. Induction of a P95/NBS1‐mediated S phase checkpoint by telomere 3′ overhang specific DNA. FASEB J. 17, 152–162 (2003)

Skin aging and photoaging: The role of DNA damage and repair

Both genetic (intrinsic) and environmental (extrinsic) factors contribute to the phenotypic changes in cutaneous aging. However, only recently have the underlying molecular mechanisms involved in these changes been elucidated. DNA damage to both genomic and mitochondrial DNA and subsequent DNA repair contribute greatly to age-associated skin changes and carcinogenesis. Better understanding of these intricate, interwoven mechanisms involved in DNA damage and repair might help to develop new strategies in preventing and treating changes of intrinsic skin aging and photoaging, improving skin appearance and reducing the risk of skin cancer.