Cortactin underpins CD44-promoted invasion and adhesion of breast cancer cells to bone marrow endothelial cells.

Using a validated tetracycline (tet)-regulated MCF7-founder (MCF7F) expression system to modulate expression of CD44 standard form (CD44s), we report the functional importance of CD44s and that of a novel transcriptional target of hyaluronan (HA)/CD44s signaling, EMS1/cortactin, in underpinning breast cancer metastasis. In functional experiments, tet-regulated induction of CD44s potentiated the migration and invasion of MCF7F cells through HA-supplemented Matrigel. EMS1/cortactin was identified by expression profiling as a novel transcriptional target of HA/CD44 signaling, an association validated by quantitative PCR and immunoblotting experiments in a range of breast cancer cell lines. The mechanistic basis underpinning CD44-promoted transcription of EMS1/cortactin was shown to be dependent upon a NFkappaB mechanism, since pharmacological inhibition of IkappaKinase-2 or suppression of p65 Rel A expression attenuated CD44-induced increases in cortactin mRNA transcript levels. Overexpression of a c-myc tagged murine cortactin construct in the weakly invasive, CD44-deficient MCF7F and T47D cells potentiated their invasion. Furthermore, the functional importance of cortactin to CD44s-promoted metastasis was demonstrated by selective suppression of cortactin in CD44-expressing MCF7F-B5 and MDA-MB-231 breast cancer cells using RNAi, which was shown to result in attenuated CD44-promoted invasion and CD44-promoted adhesion to bone marrow endothelial cells (BMECs).

The effect of ultra violet B (TL-01) phototherapy on epidermal expression of p53 protein in psoriatic plaques.

BACKGROUND: Psoriasis is a genetically determined inflammatory skin disease. It is now recognized that narrow band TL-01 phototherapy is an effective treatment for psoriasis. However, ultraviolet (UV) exposure induces p53 mutations in keratinocytes and repeated exposure of skin to UV radiation results in clonal expansion of these initiated p53-mutant cells within the epidermis. AIM: The present study aims to examine epidermal p53 expression in the skin of psoriatic patients at different time points following TL-01 phototherapy. METHODS: Skin samples from patients suffering from plaque-type psoriasis, collected before, during and at the final stages of TL-01 phototherapy were examined for p53 expression by immunohistochemistry. RESULTS/CONCLUSION: Our results showed an increase in p53 expressing keratinocytes following TL-01 phototherapy. Some of these cells were arranged spatially, as conical clones arising from putative stem cell compartments, suggesting that the chronic TL-01 treatment might have triggered cell growth and clonal expansion, an important step in initiating skin carcinogenesis.

UV irradiation augments lymphoid malignancies in mice with one functional copy of wild-type p53.

Epidemiological studies have suggested an association between exposure to solar UV radiation and the incidence of lymphoid malignancies, which has increased substantially worldwide during the last two decades. Findings from animal studies have raised the question of whether UV radiation might influence the development of lymphoid malignancies by means of its immunosuppressive effect. In this study, we examined the effect of UV irradiation on the development of lymphoid malignancies in mice with no or only one functional copy of p53. Mice that lack both copies of p53 spontaneously develop high frequency of lymphoid malignancies in the thymus and spleen. p53 heterozygous mice with only one copy of the wild-type allele also develop lymphoid malignancies, but with a much lower frequency and a long latent period. In our study using mice of the C57BL/6 background, only one of the unirradiated mice lacking one copy of p53 (p53(+/-)) spontaneously developed a lymphoid tumor (6%), whereas 88% of UV-irradiated p53(+/-) mice developed lymphoid tumors in the spleen or liver. None of the control or UV-irradiated p53 wild-type mice developed lymphoid tumors during the 60-week observation period. Both UV-irradiated and unirradiated mice lacking both copies of p53 (p53(-/-)) rapidly developed thymic lymphomas and/or lymphoid tumors in spleen or liver. All of the lymphoid tumors tested were of T cell type. The immune responses of the mice to contact sensitization were identical and were suppressed to the same extent by UV irradiation regardless of the genotype. These results indicate that differences in immune reactivity do not account for the different effects of UV radiation on lymphoid malignancies and, in addition, that p53 is not required for generation of T cell-mediated immunity. Interestingly, whereas p53 mutations or loss of heterozygosity did not account for the accelerated development of lymphoid tumors in UV-irradiated p53(+/-) mice, deletions in the p16(INK4a) gene were quite common. These data provide the experimental evidence that UV irradiation induces lymphoid neoplasms in genetically susceptible mice and support the hypothesis that extensive sunlight exposure contributes to the induction of lymphoma in humans.

Loss of Fas-ligand expression in mouse keratinocytes during UV carcinogenesis.

Skin cells containing excessive ultraviolet (UV) radiation-induced DNA damage are eliminated by apoptosis that involves the p53 pathway and Fas/Fas-Ligand (Fas-L) interactions. To determine whether dysregulation of apoptosis plays a role in skin cancer development through disruption of Fas/Fas-L interactions, hairless SKH-hr1 mice were exposed to chronic UV irradiation from Kodacel-filtered FS40 lamps for 30 weeks. Their skin was analyzed for the presence of sunburn cells (apoptotic keratinocytes) and for Fas and Fas-L expression at various time points. A dramatic decrease in the numbers of morphologically identified sunburn cells and TUNEL-positive cells was detected as early as 1 week after chronic UV exposure began. After 4 weeks of chronic UV exposure, these cells were barely detectable. This defect in apoptosis was paralleled by an initial decrease in Fas-L expression during the first week of chronic UV irradiation and a complete loss of expression after 4 weeks. Fas expression, however, increased during the course of chronic UV exposure. p53 mutations were detected in the UV-irradiated epidermis as early as 1 week after irradiation began and continued to accumulate with further UV exposure. Mice exposed to chronic UV began to develop skin tumors after approximately 8 weeks, and all mice had multiple skin tumors by 24 weeks. Most of the tumors expressed Fas but not Fas-L. We conclude that chronic UV exposure may induce a loss of Fas-L expression and a gain in p53 mutations, leading to dysregulation of apoptosis, expansion of mutated keratinocytes, and initiation of skin cancer.

Temporal events in skin injury and the early adaptive responses in ultraviolet-irradiated mouse skin.

We examined the effects of ultraviolet (UV) radiation on the time course for induction of sunburn (apoptotic) cells and expression of proteins known to be associated with growth arrest and apoptosis in SKH-hr1 mouse skin. Mice were irradiated with a single dose (2.5 kJ/m(2)) of UV from Kodacel-filtered (290-400 nm) FS40 sunlamps and the skin tissues were analyzed at various times after irradiation for the presence of apoptotic cells and expression of p53, p21(Waf-1/Cip1), bcl-2, bax, and proliferating cell nuclear antigen. The results indicated that p53 expression was induced early in the epidermis, reaching maximum levels 12 hours after irradiaton, and p21(Waf-1/Cip1) expression in the epidermis peaked at 24 hours after irradiation. In contrast, UV radiation induced high levels of bax at 24 to 72 hours after irradiation with a concomitant decrease in bcl-2 expression. Coinciding with these changes, apoptotic cells began to appear 6 hours after irradiation and reached a maximum at 24 hours after irradiation. Interestingly, proliferating cell nuclear antigen expression, which was initially confined to the basal layer, became dispersed throughout the basal and suprabasal layers of the skin at 48 hours and paralleled marked hyperplasia. These results suggest that UV irradiation of mouse skin induces apoptosis mediated by the p53/p21/bax/bcl-2 pathway and that the dead cells are replaced by hyperproliferative cells, leading to epidermal hyperplasia. This implies that UV-induced apoptosis and hyperplasia are closely linked and tightly regulated and that dysregulation of these two events may lead to skin cancer development.

Persistence of p53 mutations and resistance of keratinocytes to apoptosis are associated with the increased susceptibility of mice lacking the XPC gene to UV carcinogenesis.

Like xeroderma pigmentosum (XP) patients, transgenic mice lacking nucleotide excision repair (NER) genes such as XPA and XPC are extremely susceptible to ultraviolet (UV)-induced skin cancer. Because the p53 gene is an important target for UV carcinogenesis and because the p53 protein modulates NER, we investigated the consequences of NER deficiency on UV-induced p53 mutations in XPC-/- mouse skin tumors. Thirty-eight (76%) of 50 UV-induced XPC-/- skin tumor analysed displayed C-->T or CC-->TT transitions at dipyrimidine sites on the untranscribed strand of the p53 gene. A major hot spot for p53 mutation occurred at codon 270, which is also a hot spot in UV-induced skin tumors from NER-proficient C3H and SKH-hr 1 mice. Interestingly, codon 270 mutations were induced in both XPC-/- and +/+ mouse skin after 1 week of UV irradiation, but the mutations persisted only in XPC-/- mouse skin after 3 - 4 weeks of chronic UV. The persistence of UV-induced p53 mutations in XPC-/- mouse skin was associated with decreased apoptosis and increased proliferation of keratinocytes, suggesting that these events may contribute to the accelerated development of UV-induced skin tumors in XPC-/- mice.

Fas ligand: a sensor for DNA damage critical in skin cancer etiology.

DNA-damaged cells can either repair the DNA or be eliminated through a homeostatic control mechanism termed "cellular proofreading." Elimination of DNA-damaged cells after ultraviolet radiation (UVR) through sunburn cell (apoptotic keratinocyte) formation is thought to be pivotal for the removal of precancerous skin cells. Sunburn cell formation was found to be dependent on Fas ligand (FasL), a pro-apoptotic protein induced by DNA damage. Chronic exposure to UVR caused 14 of 20 (70 percent) FasL-deficient mice and 1 of 20 (5 percent) wild-type mice to accumulate p53 mutations in the epidermis. Thus, FasL-mediated apoptosis is important for skin homeostasis, suggesting that the dysregulation of Fas-FasL interactions may be central to the development of skin cancer.

UV-radiation-specific p53 mutation frequency in normal skin as a predictor of risk of basal cell carcinoma.

BACKGROUND: A strong association has been found between skin cancer and exposure to UV radiation. The p53 tumor suppressor gene (also known as TP53), which is frequently mutated in human cancers, is believed to be an early target in UV radiation-associated skin carcinogenesis. We have previously developed a sensitive, polymerase chain reaction-based method capable of detecting and quantifying a UV radiation-specific mutation in the p53 gene (codons 247 and 248: AAC CGG --> AAT TGG) in normal skin. We have used this method to examine whether UV radiation-specific mutation frequency is associated with risk of basal cell carcinoma (BCC) and with sun exposure. METHODS: This case-control study in Australia involved 53 case subjects with BCC and 75 control subjects. DNA was isolated from normal skin (mirror-image anatomic site to the cancer site for case subjects and a randomly selected site for control subjects) and assayed for p53 mutation. Relationships between p53 mutation frequency and risk of BCC, sun sensitivity, or sun exposure were estimated by use of odds ratios (ORs) and 95% confidence intervals (95% CIs). RESULTS: Case subjects were more likely to have a p53 mutation than control subjects (OR = 3.1; 95% CI = 1.3-7.1). In addition, the odds of BCC increased monotonically with increasing frequency of p53 mutation. No statistically significant associations could be demonstrated between p53 mutation frequency and age, sex, sensitivity to the sun, pigmentary characteristics, total lifetime sun exposure, or sun exposure to the biopsy site. CONCLUSIONS: Our results indicate that tandem CC --> TT mutations involving codons 247 and 248 of the p53 gene are associated with an increased risk of BCC but cannot be used as an accurate measure of total UV-radiation exposure.

Mechanisms of induction of skin cancer by UV radiation.

Ultraviolet (UV) radiation is the carcinogenic factor in sunlight; damage to skin cells from repeated exposure can lead to the development of cancer. UV radiation has been mainly implicated as the cause of non-melanoma skin cancer, although some role for UV in malignant melanoma has been suggested. The induction of skin cancer is mainly caused by the accumulation of mutations caused by UV damage. Cellular mechanisms exist to repair the DNA damage, or to induce apoptosis to remove severely damaged cells; however, the additive effects of mutations in genes involved in these mechanisms, or in control of the cell cycle, can lead to abnormal cell proliferation and tumor development. The molecular events in the induction of skin cancer are being actively investigated, and recent research has added to the understanding of the roles of tumor suppressor and oncogenes in skin cancer. UV radiation has been shown to induce the expression of the p53 tumor suppressor gene, and is known to produce "signature" mutations in p53 in human and mouse skin cancers and in the tumor suppressor gene patched in human basal cell carcinoma. The role of UV radiation in suppression of immune surveillance in the skin, which is an important protection against skin tumor development, is also being investigated. The knowledge gained will help to better understand the ways in which skin cancer arises from UV exposure, which will in turn allow development of better methods of treatment and prevention.

Quantitative detection of ultraviolet-specific p53 mutations in normal skin from Japanese patients.

We have previously developed sensitive methods to detect UV-specific p53 mutations (CC to TT tandem mutations) and have reported that such mutations could be found in the normal skin cell populations of sun-exposed body sites, but not in those of covered sites, in Australian cancer patients. We have now further refined our allele-specific PCR method for detecting CC to TT mutations at codons 247/248 of the p53 gene to allow quantitative measurements. Using DNA containing this mutation from a tumor as a standard for calibration and 5 micrograms of genomic DNA/PCR reaction, we could detect 1 mutant allele in about 10(6) wild-type alleles. It is essential to use purified primers and 64 degrees C as the annealing temperature for PCR. Our method has been applied in a study of the correlation of sun exposure and accumulation of CC to TT mutations in normal skin biopsies from Japanese patients. There were more p53 mutations in samples taken from sites that were chronically exposed to the sun than in those from covered sites. A significant trend of increased p53 mutation frequency with increase in age of subjects was found, suggesting the cumulative nature of the mutation. On the other hand, the p53 mutation frequency was higher in patients with premalignant tumors or nonmelanocytic skin cancer than in patients with only benign tumors. These results confirm the utility of PCR-based p53 gene mutation assays for the measurement of exposure to UV as well as for predicting the risk of UV-associated skin cancer.

Evidence for UV-associated activation of telomerase in human skin.

Telomerase activation plays a crucial role in the immortalization of human cells and carcinogenesis; however, the temporal and pathophysiological aspects of the activation in vivo are poorly understood. We found telomerase activity not only in malignant tumors (91%) but also in most benign (60%) and premalignant (89%) skin tumors. This suggests the involvement of telomerase activation in a crucial biological step of human skin carcinogenesis. Because UV light is a major factor in skin carcinogenesis, we further examined telomerase activity in normal skin samples and in normal skin samples adjacent to benign, premalignant, and malignant skin lesions. Data for chronically sun-exposed body sites were compared with those for covered sites. Among normal skin samples, 39% (26 of 67) had telomerase activity, and this activity was unrelated to neighboring lesions but strongly associated with the level of sun exposure. Fifty-four % (21 of 39) of normal skin samples from chronically sun-exposed sites were telomerase-positive, compared with only 12% (3 of 26) of samples from covered sites. When we examined telomerase activity and CC to TT mutations at codons 247/8 of the p53 gene (which are considered to be UV specific) in the same normal skin samples, only 43% (7 of 16) of telomerase-positive normal skin samples at sun-exposed sites contained the p53 mutations, whereas all (7 of 7) of the samples with UV-specific p53 mutations showed telomerase activity (P = 0.019). These data suggest that telomerase activation is involved at an early stage of human skin carcinogenesis and that activation may precede the acquisition of UV-associated p53 mutations in the skin. Telomerase activity was also found in plucked hair follicles and enzymatically separated epidermis, which may be associated with the presence of stem cells in the skin.

Prolactin receptor immunoreactivity in rat anterior pituitary.

Prolactin (PRL) receptors have been identified in both classical and nonclassical target organs. Actions of PRL on the secretion of anterior pituitary hormones have been reported. In order to confirm the presence of PRL receptors on specific cell types of the anterior pituitary, immunocytology on ultrathin frozen sections was carried out using monoclonal antibodies specific to the rat PRL receptor. Anterior pituitary glands were removed and fixed in 4% paraformaldehyde or 2.5% glutaraldehyde and postfixed in 1% osmium tetroxide. Tissues were frozen and ultrathin sections were made by cryoultramicrotomy. In anterior pituitary cells, all endocrine cell populations showed PRL-R-like immunoreactivity. The relative labeling frequency based on immunolabeling studies was somatotrophs > lactotrophs > thyrotrophs approximately corticotrophs > gonadotrophs. Within all endocrine cell types, no apparent differences could be observed in the subcellular localization of PRL-R-like immunoreactivity. At the subcellular level, gold particles, either isolated or in groups of two, were seen at the level of the plasma membrane, in the cytoplasmic matrix, in the vicinity of the endoplasmic reticulum and in the nucleus. The identification of receptors specific to PRL within different cells of the anterior pituitary raises the interesting possibility that PRL may act via an autocrine or paracrine network in regulating anterior pituitary function.