AMF1 (GPS2) modulates p53 transactivation.

We have reported that the papillomavirus E2 protein binds the nuclear factor AMF1 (also called G-protein pathway suppressor 2 or GPS2) and that their interaction is necessary for transcriptional activation by E2. It has also been shown that AMF1 can influence the activity of cellular transcription factors. These observations led us to test whether AMF1 regulates the functions of p53, a critical transcriptional activator that integrates stress signals and regulates cell cycle and programmed cell death. We report that AMF1 associates with p53 in vivo and in vitro and facilitates the p53 response by augmenting p53-dependent transcription. Overexpression of AMF1 in U2OS cells increases basal level p21(WAF1/CIP1) expression and causes a G(1) arrest. U2OS cells stably overexpressing AMF1 show increased apoptosis upon exposure to UV irradiation. These data demonstrate that AMF1 modulates p53 activities.

Multiple functions of human papillomavirus type 16 E6 contribute to the immortalization of mammary epithelial cells.

The E6 proteins from cervical cancer-associated human papillomavirus (HPV) types such as HPV type 16 (HPV-16) induce proteolysis of the p53 tumor suppressor protein through interaction with E6-AP. We have previously shown that human mammary epithelial cells (MECs) immortalized by HPV-16 E6 display low levels of p53. HPV-16 E6 as well as other cancer-related papillomavirus E6 proteins also binds the cellular protein E6BP (ERC-55). To explore the potential functional significance of these interactions, we created and analyzed a series of E6 mutants for their ability to interact with E6-AP, p53, and E6BP in vitro. While there was a similar pattern of binding among these E6 targets, a subset of mutants differentiated E6-AP binding, p53 binding, and p53 degradation activities. These results demonstrated that E6 binding to E6-AP is not sufficient for binding to p53 and that E6 binding to p53 is not sufficient for inducing p53 degradation. The in vivo activity of these HPV-16 E6 mutants was tested in MECs. In agreement with the in vitro results, most of these p53 degradation-defective E6 mutants were unable to reduce the p53 level in early-passage MECs. Interestingly, several mutants that showed severely reduced ability for interacting with E6-AP, p53, and E6BP in vitro efficiently immortalized MECs. These immortalized cells exhibited low p53 levels at late passage. Furthermore, mutants defective for p53 degradation but able to immortalize MECs were also identified, and the immortal cells retained normal levels of p53 protein. These results imply that multiple functions of HPV-16 E6 contribute to MEC immortalization.

The domain of p53 required for binding HPV 16 E6 is separable from the degradation domain.

The E6 proteins of specific cancer-associated human papillomaviruses (HPVs) complex with and mediate degradation of the cellular anti-oncogene p53 in vitro. A critical property of p53 is its ability to stimulate transcription from promoters containing its recognition sequence. HPV E6, mutant p53 proteins, and several DNA tumor virus oncogenes inhibit the transcriptional activity of wild-type p53. In this report, the structural requirements for the interaction between HPV 16 E6 and p53 were examined both in vivo and in vitro. p53-stimulated transcription was efficiently inhibited by wild-type HPV 16 E6 and E6 mutants competent for p53 binding and degradation. A series of p53 deletions and hybrid proteins with heterologous DNA binding, dimerization and transactivation domains were analysed for transcriptional interaction with HPV 16 E6 to determine the domains of p53 required for transcriptional inhibition. These chimeric proteins were also analysed for E6 binding and E6-mediated degradation in vitro. In both assays, complex formation with E6 was mediated through the amino-terminal 345 amino acids of p53 without a specific requirement for its C-terminus. Hybrid proteins containing residues 161-345 of p53 also bound E6, but this segment of p53 was not susceptible to E6 induced proteolysis. A second region of p53, within its N-terminal 160 aa, is required for E6 induced degradation of complexed p53. Taken together, these results suggest that the complex formation between E6 and p53 is not mediated through the C-terminus of p53 and that binding and degradation are separable.

Regulation of human melanocyte growth, dendricity, and melanization by keratinocyte derived factors.

Epidermal pigmentation involves the synthesis of melanin in melanocytes and its transfer to surrounding keratinocytes, where it functions in photoprotection. To investigate the possible role of the keratinocyte in regulating pigmentation, human keratinocytes were incubated for 24 h in a defined culture medium, which was then transferred to pure human melanocyte cultures. After 1 week, the conditioned medium produced a fourfold increase in melanocyte yield and a seven-fold increase in total melanin. Increased melanocyte dendricity was clearly visible within 24 h as well. Ultrafiltration of the keratinocyte-conditioned medium suggested approximately one-half of the growth promoting activity as well as most of the dendricity and melanization stimulating activities were of low molecular weight (less than 500 Da). High molecular weight fractions stimulated only melanocyte growth. Of the several known keratinocyte-derived factors tested, none could be implicated as a mediator of the observed effects. Basic fibroblast growth factor, known to stimulate melanocyte growth in some culture systems, failed to stimulate growth, dendricity, or melanin content when added to the complete non-conditioned medium. Interleukin-1 alpha, interleukin-1 beta, 12-hydroxyeicosatetraenoic acid, prostaglandin E2, leukotriene B4, and adenosine 3',5'-cyclic monophosphate analogues also had no effect. These studies demonstrate that keratinocytes in vitro release factors that modulate melanocyte behavior and expand our understanding of controls for human epidermal pigmentation.

Induction of nerve growth factor receptors on cultured human melanocytes.

Normal differentiation and malignant transformation of human melanocytes involve a complex series of interactions during which both genetic and environmental factors play roles. At present, the regulation of these processes is poorly understood. We have induced the expression of nerve growth factor (NGF) receptors on cultured human melanocytes with phorbol 12-tetradecanoate 13-acetate and have correlated this event with the appearance of a more differentiated, dendritic morphology. Criteria for NGF receptor expression included protein accumulation and cell-surface immunofluorescent staining with a monoclonal antibody directed against the human receptor and induction of the messenger RNA species as determined by blot-hybridization studies. The presence of the receptor could also be induced by UV irradiation or growth factor deprivation. The NGF receptor is inducible in cultured human melanocytes, and we suggest that NGF may modulate the behavior of this neural crest-derived cell in the skin.

Vitamin D, its precursors, and metabolites do not affect melanization of cultured human melanocytes.

Exposure of the skin to sunlight results in both tanning and vitamin D3 production. It has therefore been suggested that vitamin D3 or its active metabolite 1,25-dihydroxyvitamin D3 may be the mediator of UV-induced melanogenesis. To test this hypothesis, newborn foreskin-derived melanocytes were cultured in paired dishes in hormone-supplemented medium with 2% serum containing no detectable vitamin D3 or in the same medium containing 10(-8) or 10(-10) M of either provitamin D3, lumisterol, previtamin D3, vitamin D3, 25-hydroxyvitamin D3, or 1,25-dihydroxyvitamin D3. After 10 days, cell number in cultures containing vitamin D compounds was 93%-140% of unsupplemented controls and melanin content was 60%-120% of control, with no significant difference in either parameter for any compound tested. In separate experiments, human melanocytes and Cloudman S91 melanoma cells were repeatedly irradiated with physiologic doses of simulated sunlight and incubated between irradiations with provitamin D3, previtamin D3, vitamin D3, or 1,25-dihydroxyvitamin D3. Irradiated cultures had a 90%-95% inhibition of cell growth associated with a 200%-800% increase in melanin content per cell relative to controls, but there was no effect of any vitamin D compound on either cell type. Neither cultured human melanocytes nor S91 cells showed evidence of the cytosolic 1,25-dihydroxy-vitamin D3 receptor binding by sucrose density gradient analysis using radiolabeled 1,25-dihydroxyvitamin D3. The combined data strongly suggest that neither vitamin D3 nor its precursors or metabolites directly mediate melanogenesis in these cells.