An analysis of iris pattern as a risk factor for skin cancer development in immunosuppressed renal transplant recipients.

INTRODUCTION: Renal transplant recipients are at increased risk of keratinocyte skin cancers with a tendency to have multiple, aggressive and difficult to treat tumours. The eye and the skin share the same embryological ectoderm. Iris pattern has recently been reported as a predictive risk factor for skin cancer in non-immunosuppressed Southern European (Grigore et al., J Eur Acad Dermatol Venereol, 2018, 1662) and Irish populations (Ridge et al., J Eur Acad Dermatol Venereol, 2022, e542). AIMS: To analyse if an individual's iris pattern is an independent risk factor for the development of keratinocyte skin cancers in renal transplant recipients. METHODS: Iris patterns of 110 renal transplant recipients were evaluated using the Simionescu visual three-step technique (iris periphery, colarette and iris freckling [Simionescu et al., Ann Res Rev Biol, 2014, 2525]). Established risk factors for skin cancer in transplant patients were recorded as confounding factors. RESULTS: Observational cross-sectional study including 110 renal transplant population. Thirty-one participants had skin cancer. In the skin cancer group, iris periphery was blue/grey in 74.3% (p = 0.053, OR 2.5), the colarette was light brown in 57.1% (p < 0.0043) and iris freckles were present in 55%(p = 0.044). Dark brown and blue colarettes were observed in controls. Binary Logistic Regression analysis showed light brown colarette is a significant independent risk factor for skin cancer (OR 4.54, p < 0.02, CI 1.56-10.57). CONCLUSION: Within this renal transplant population a blue iris periphery, light brown colarette and presence of freckling confers an independent risk for keratinocyte skin cancer. Iris pattern is a useful tool for identification of transplant patients at risk of keratinocyte skin cancer and an easy-to-use technique for risk evaluation in this cohort. This is the first study looking at iris pattern and keratinocyte skin cancer risk in renal transplant population.

Actin' up: RhoB in cancer and apoptosis.

RhoB is a small GTPase that regulates actin organization and vesicle transport. It is required for signalling apoptosis in transformed cells that are exposed to farnesyltransferase inhibitors, DNA-damaging agents or taxol. Genetic analysis in mice indicates that RhoB is dispensable for normal cell physiology, but that it has a suppressor or negative modifier function in stress-associated processes, including cancer.

BIN1 is a novel MYC-interacting protein with features of a tumour suppressor.

BIN1 is a novel protein that interacts with the functionally critical Myc box regions at the N terminus of the MYC oncoprotein. BIN1 is structurally related to amphiphysin, a breast cancer-associated autoimmune antigen, and RVS167, a negative regulator of the yeast cell cycle, suggesting roles in malignancy and cell cycle control. Consistent with this likelihood, BIN1 inhibited malignant cell transformation by MYC. Although BIN1 is expressed in many normal cells, its levels were greatly reduced or undetectable in 14/27 carcinoma cell lines and 3/6 primary breast tumours. Deficits were functionally significant because ectopic expression of BIN1 inhibited the growth of tumour cells lacking endogenous message. We conclude that BIN1 is an MYC-interacting protein with features of a tumour suppressor.

Farnesyltransferase inhibitors: antineoplastic mechanism and clinical prospects.

Recent work suggests that farnesyltransferase inhibitors suppress cancer cell proliferation through mechanisms other than inhibiting Ras isoprenylation, which is not a crucial event. Recent evidence also suggests that the antineoplastic properties of farnesyltransferase inhibitors are due to alterations in the isoprenylation of RhoB, an endosomal Rho protein that functions in receptor trafficking. A shift in conceptual focus from Ras to Rho to understand how farnesyltransferase inhibitors act provides a new vantage to address old questions in the field and suggests strategies to improve and potentially widen clinical applications.

Methylation-sensitive sequence-specific DNA binding by the c-Myc basic region.

The function of the c-Myc oncoprotein and its role in cell growth control is unclear. A basic region of c-Myc is structurally related to the basic motifs of helix-loop-helix (HLH) and leucine zipper proteins, which provide sequence-specific DNA binding function. The c-Myc basic region was tested for its ability to bind DNA by attaching it to the HLH dimerization interface of the E12 enhancer binding factor. Dimers of the chimeric protein, termed E6, specifically bound an E box element (GGCCACGTGACC) recognized by other HLH proteins in a manner dependent on the integrity of the c-Myc basic motif. Methylation of the core CpG in the E box recognition site specifically inhibited binding by E6, but not by two other HLH proteins. Expression of E6 (but not an E6 DNA binding mutant) suppressed the ability of c-myc to cooperate with H-ras in a rat embryo fibroblast transformation assay, suggesting that the DNA recognition specificity of E6 is related to that of c-Myc in vivo.

RhoB facilitates c-Myc turnover by supporting efficient nuclear accumulation of GSK-3.

The small GTPase RhoB suppresses cancer in part by limiting cell proliferation. However, the mechanisms it uses to achieve this are poorly understood. Recent studies link RhoB to trafficking of Akt, which through its regulation of glycogen synthase kinase-3 (GSK-3) has an important role in controlling the stability of the c-Myc oncoprotein. c-Myc stabilization may be a root feature of human tumorigenesis as it phenocopies an essential contribution of SV40 small T antigen in human cell transformation. In this study we show that RhoB directs efficient turnover of c-Myc in established or transformed mouse fibroblasts and that the attenuation of RhoB which occurs commonly in human cancer is a sufficient cause to elevate c-Myc levels. Increased levels of c-Myc elicited by RhoB deletion increased the proliferation of nullizygous cells, whereas restoring RhoB in null cells decreased the stability of c-Myc and restrained cell proliferation. Mechanistic analyses indicated that RhoB facilitated nuclear accumulation of GSK-3 and GSK-3-mediated phosphorylation of c-Myc T58, the critical site for ubiquitination and degradation of c-Myc. RhoB deletion restricted nuclear localization of GSK-3, reduced T58 phosphorylation, and stabilized c-Myc. These effects were not associated with changes in phosphorylation or localization of Akt, however, differences were observed in phosphorylation and localization of the GSK-3 regulatory Akt-related kinase, serum- and glucocorticoid-inducible protein kinase (SGK). The ability of RhoB to support GSK-3-dependent turnover of c-Myc offers a mechanism by which RhoB acts to limit the proliferation of neoplastically transformed cells.

A new bind for Myc.

Recent studies centered on the c-Myc basic/helix-loop-helix/leucine zipper (B/HLH/LZ) motifs have led to the identification of a DNA recognition sequence for c-Myc and the isolation of a novel protein that forms a DNA-binding complex with c-Myc in vitro. These advances may make it possible to address directly the long-standing question of c-Myc function in vivo.

Plasminogen activator inhibitor-1 gene-deficient mice. I. Generation by homologous recombination and characterization.

Homozygous plasminogen activator inhibitor-1 (PAI-1)-deficient (PAI-1-/-) mice were generated by homologous recombination in D3 embryonic stem cells. Deletion of the genomic sequences encompassing the transcription initiation site and the entire coding regions of murine PAI-1 was demonstrated by Southern blot analysis. A 3.0-kb PAI-1-specific mRNA was identified by Northern blot analysis in liver from PAI-1 wild type (PAI-1+/+) but not from PAI-1-/- mice. Plasma PAI-1 levels, measured 2-4 h after endotoxin (2.0 mg/kg) injection were 63 +/- 2 ng/ml, 30 +/- 10 ng/ml, and undetectable (< 2 ng/ml) in PAI-1+/+, heterozygous (PAI-1+/-) and PAI-1-/- mice, respectively (mean +/- SEM, n = 4-11). PAI-1-specific immunoreactivity was demonstrable in kidneys of PAI-1+/+ but not of PAI-1-/- mice. SDS-gel electrophoresis of plasma incubated with 125I-labeled recombinant human tissue-type plasminogen activator revealed an approximately 115,000-M(r) component with plasma from endotoxin-stimulated (0.5 mg/kg) PAI-1+/+ but not from PAI-1-/- mice, which could be precipitated with a polyclonal anti-PAI-1 antiserum. PAI-1-/- mice were viable, produced similar sizes of litters as PAI-1+/+ mice, and showed no apparent macroscopic or microscopic histological abnormalities.

Posttranscriptional regulation of cellular gene expression by the c-myc oncogene.

The c-myc oncogene has been implicated in the development of many different cancers, yet the mechanism by which the c-myc protein alters cellular growth control has proven elusive. We used a cDNA hybridization difference assay to isolate two genes, mr1 and mr2, that were constitutively expressed (i.e., deregulated) in rodent fibroblast cell lines immortalized by transfection of a viral promoter-linked c-myc gene. Both cDNAs were serum inducible in quiescent G0 fibroblasts, suggesting that they are functionally related to cellular proliferative processes. Although there were significant differences in cytoplasmic mRNA levels between myc-immortalized and control cells, the rates of transcription and mRNA turnover of both genes were similar, suggesting that c-myc regulates mr1 and mr2 expression by some nuclear posttranscriptional mechanism. mr1 was also rapidly (within 2 h) and specifically induced by dexamethasone in BALB/c cell lines expressing a mouse mammary tumor virus long terminal repeat-driven myc gene, under conditions where other growth factor-inducible genes were unaffected. A frameshift mutation in the mouse mammary tumor virus myc gene destroyed the dexamethasone stimulation of mr1, indicating that c-myc protein is required for the effect. As in the myc-immortalized cells, the induction of mr1 by c-myc occurred without detectable changes in mr1 transcription or cytoplasmic mRNA stability, implicating regulation, either direct or indirect, through a nuclear posttranscriptional mechanism. These results provide evidence that c-myc can rapidly modulate cellular gene expression and suggest that c-myc may function in gene regulation at the level of RNA export, splicing, or nuclear RNA turnover.

Evidence that farnesyltransferase inhibitors suppress Ras transformation by interfering with Rho activity.

Small-molecule inhibitors of the housekeeping enzyme farnesyltransferase (FT) suppress the malignant growth of Ras-transformed cells. Previous work suggested that the activity of these compounds reflected effects on actin stress fiber regulation rather than Ras inhibition. Rho proteins regulate stress fiber formation, and one member of this family, RhoB, is farnesylated in vivo. Therefore, we tested the hypothesis that interference with RhoB was the principal basis by which the peptidomimetic FT inhibitor L-739,749 suppressed Ras transformation. The half-life of RhoB was found to be approximately 2 h, supporting the possibility that it could be functionally depleted within the 18-h period required by L-739,749 to induce reversion. Cell treatment with L-739,749 disrupted the vesicular localization of RhoB but did not effect the localization of the closely related RhoA protein. Ras-transformed Rat1 cells ectopically expressing N-myristylated forms of RhoB (Myr-rhoB), whose vesicular localization was unaffected by L-739,749, were resistant to drug treatment. The protective effect of Myr-rhoB required the integrity of the RhoB effector domain and was not due to a gain-of-function effect of myristylation on cell growth. In contrast, Rat1 cells transformed by a myristylated Ras construct remained susceptible to growth inhibition by L-739,749. We concluded that Rho is necessary for Ras transformation and that FT inhibitors suppress the transformed phenotype at least in part by direct or indirect interference with Rho, possibly with RhoB itself.

Cell growth inhibition by farnesyltransferase inhibitors is mediated by gain of geranylgeranylated RhoB.

Recent results have shown that the ability of farnesyltransferase inhibitors (FTIs) to inhibit malignant cell transformation and Ras prenylation can be separated. We proposed previously that farnesylated Rho proteins are important targets for alternation by FTIs, based on studies of RhoB (the FTI-Rho hypothesis). Cells treated with FTIs exhibit a loss of farnesylated RhoB but a gain of geranylgeranylated RhoB (RhoB-GG), which is associated with loss of growth-promoting activity. In this study, we tested whether the gain of RhoB-GG elicited by FTI treatment was sufficient to mediate FTI-induced cell growth inhibition. In support of this hypothesis, when expressed in Ras-transformed cells RhoB-GG induced phenotypic reversion, cell growth inhibition, and activation of the cell cycle kinase inhibitor p21WAF1. RhoB-GG did not affect the phenotype or growth of normal cells. These effects were similar to FTI treatment insofar as they were all induced in transformed cells but not in normal cells. RhoB-GG did not promote anoikis of Ras-transformed cells, implying that this response to FTIs involves loss-of-function effects. Our findings corroborate the FTI-Rho hypothesis and demonstrate that gain-of-function effects on Rho are part of the drug mechanism. Gain of RhoB-GG may explain how FTIs inhibit the growth of human tumor cells that lack Ras mutations.

The c-myc-regulated gene mrl encodes plasminogen activator inhibitor 1.

The DNA sequence of the c-myc-regulated gene mrl (G. C. Prendergast and M. D. Cole, Mol. Cell. Biol. 9:124-134, 1989) reveals that it encodes plasminogen activator inhibitor 1 (PAI-1), a regulator of extracellular proteolysis. Comparison of the human and mouse PAI-1 promoters and cDNA 3' noncoding regions revealed several highly conserved sequence domains, potential targets for c-myc and other factors influencing PAI-1 expression. We discuss possible roles for PAI-1 in normal and neoplastic cell growth control.

Activated H-ras rescues E1A-induced apoptosis and cooperates with E1A to overcome p53-dependent growth arrest.

The adenovirus E1A oncogene products stimulate DNA synthesis and cell proliferation but fail to transform primary baby rat kidney (BRK) cells because of the induction of p53-mediated programmed cell death (apoptosis). Overexpression of dominant mutant p53 (to abrogate wild-type p53 function) or introduction of apoptosis inhibitors, such as adenovirus E1B 19K or Bcl-2 oncoproteins, prevents E1A-induced apoptosis and permits transformation of BRK cells. The ability of activated Harvey-ras (H-ras) to cooperate with E1A to transform BRK cells suggests that H-ras is capable of overcoming the E1A-induced, p53-dependent apoptosis. We demonstrate here that activated H-ras was capable of suppressing apoptosis induced by E1A and wild-type p53. However, unlike Bcl-2 and the E1B 19K proteins, which completely block apoptosis but not p53-dependent growth arrest, H-ras expression permitted DNA synthesis and cell proliferation in the presence of high levels of wild-type p53. The mechanism by which H-ras regulates apoptosis and cell cycle progression is thereby strikingly different from that of the E1B 19K and Bcl-2 proteins. BRK cells transformed with H-ras and the temperature sensitive murine mutant p53(val 135), which lack E1A, underwent growth arrest at the permissive temperature for wild-type p53. p53-dependent growth arrest, however, could be relieved by E1A expression. Thus, H-ras alone was insufficient and cooperation of H-ras and E1A was required to override growth suppression by p53. Our data further suggest that two complementary growth signals from E1A plus H-ras can rescue cell death and thus permit transformation.

RhoB is dispensable for mouse development, but it modifies susceptibility to tumor formation as well as cell adhesion and growth factor signaling in transformed cells.

RhoB is an endosomal small GTPase that is implicated in the response to growth factors, genotoxic stress, and farnesyltransferase inhibitors. To gain insight into its physiological functions we examined the consequences of homozygous gene deletion in the mouse. Loss of RhoB did not adversely affect mouse development, fertility, or wound healing. However, embryo fibroblasts cultured in vitro exhibited a defect in motility, suggesting that RhoB has a role in this process that is conditional on cell stress. Neoplastic transformation by adenovirus E1A and mutant Ras yielded differences in cell attachment and spreading that were not apparent in primary cells. In addition, transformed -/- cells displayed altered actin and proliferative responses to transforming growth factor beta. A negative modifier role in transformation was suggested by the increased susceptibility of -/- mice to 7,12-dimethylbenz[a]anthracene-induced skin carcinogenesis and by the increased efficiency of intraperitoneal tumor formation by -/- cells. Our findings suggest that RhoB is a negative regulator of integrin and growth factor signals that are involved in neoplastic transformation and possibly other stress or disease states.

A role for the putative tumor suppressor Bin1 in muscle cell differentiation.

Bin1 is a Myc-interacting protein with features of a tumor suppressor. The high level of Bin1 expression in skeletal muscle prompted us to investigate its role in muscle differentiation. Significant levels of Bin1 were observed in undifferentiated C2C12 myoblasts, a murine in vitro model system. Induction of differentiation by growth factor withdrawal led to an upregulation of Bin1 mRNA and to the generation of higher-molecular-weight forms of Bin1 protein by alternate splicing. While Bin1 in undifferentiated cells was localized exclusively in the nucleus, differentiation-associated isoforms of Bin1 were found in the cytoplasm as well. To examine the function of Bin1 during differentiation, we generated stable cell lines that express exogenous human Bin1 cDNA in the sense or antisense orientation. Cells overexpressing Bin1 grew more slowly than control cells and differentiated more rapidly when deprived of growth factors. In contrast, C2C12 cells expressing antisense Bin1 showed an impaired ability to undergo differentiation. Taken together, the results indicated that Bin1 expression, structure, and localization are tightly regulated during muscle differentiation and suggested that Bin1 plays a functional role in the differentiation process.

Differential effects of protein kinase A on Ras effector pathways.

Ras mutants with the ability to interact with different effectors have played a critical role in the identification of Ras-dependent signaling pathways. We used two mutants, RasS35 and RasG37, which differ in their ability to bind Raf-1, to examine Ras-dependent signaling in thyroid epithelial cells. Wistar rat thyroid cells are dependent upon thyrotropin (TSH) for growth. Although TSH-stimulated mitogenesis requires Ras, TSH activates protein kinase A (PKA) and downregulates signaling through Raf and the mitogen-activated protein kinase (MAPK) cascade. Cells expressing RasS35, a mutant which binds Raf, or RasG37, a mutant which binds RalGDS, exhibited TSH-independent proliferation. RasS35 stimulated morphological transformation and anchorage-independent growth. RasG37 stimulated proliferation but not transformation as measured by these indices. TSH exerted markedly different effects on the Ras mutants and transiently repressed MAPK phosphorylation in RasS35-expressing cells. In contrast, TSH stimulated MAPK phosphorylation and growth in cells expressing RasG37. The Ras mutants, in turn, exerted differential effects on TSH signaling. RasS35 abolished TSH-stimulated changes in cell morphology and thyroglobulin expression, while RasG37 had no effect on these activities. Together, the data indicate that cross talk between Ras and PKA discriminates between distinct Ras effector pathways.

c-Myc cooperates with activated Ras to induce the cdc2 promoter.

Expression of c-myc with constitutively active mutants of the ras gene results in the cooperative transformation of primary fibroblasts, although the precise mechanism by which these genes cooperate is unknown. Since c-Myc has been shown to function as a transcriptional activator, we have examined the ability of c-Myc and activated Ras (H-RasV-12) to cooperatively induce the promoter activity of cdc2, a gene which is critical for cell cycle progression. Microinjection of expression constructs encoding H-RasV-12 and c-Myc along with a cdc2 promoter-luciferase reporter plasmid into quiescent cells led to an increase in cdc2 promoter activity approximately 30 h after injection, a period which coincides with the S-to-G2/M transition in these cells. Expression of H-RasV-12 alone weakly activated the cdc2 promoter, while expression of c-Myc alone had no effect. Mutants of c-Myc lacking either the leucine zipper dimerization domain or the phosphoacceptor site Ser-62 could not cooperate with H-RasV-12 to induce the cdc2 promoter. These mutants also lacked the ability to cooperate with H-RasV-12 to stimulate DNA synthesis. Deletion analysis identified a distinct region of the cdc2 promoter which was required for c-Myc responsiveness. Taken together, these observations suggest a mechanistic link between the molecular activities of c-Myc and Ras and induction of the cell cycle regulator Cdc2.

Protein kinase A-dependent and -independent signaling pathways contribute to cyclic AMP-stimulated proliferation.

The effects of cyclic AMP (cAMP) on cell proliferation are cell type specific. Although the growth-inhibitory effects of cAMP have been well studied, much less is known regarding how cAMP stimulates proliferation. We report that cAMP stimulates proliferation through both protein kinase A (PKA)-dependent and PKA-independent signaling pathways and that phosphatidylinositol 3-kinase (PI3K) is required for cAMP-stimulated mitogenesis. In cells where cAMP is a mitogen, cAMP-elevating agents stimulate membrane ruffling, Akt phosphorylation, and p70 ribosomal S6 protein kinase (p70s6k) activity. cAMP effects on ruffle formation and Akt were PKA independent but sensitive to wortmannin. In contrast, cAMP-stimulated p70s6k activity was repressed by PKA inhibitors but not by wortmannin or microinjection of the N-terminal SH2 domain of the p85 regulatory subunit of PI3K, indicating that p70s6k and Akt can be regulated independently. Microinjection of highly specific inhibitors of PI3K or Rac1, or treatment with the p70s6k inhibitor rapamycin, impaired cAMP-stimulated DNA synthesis, demonstrating that PKA-dependent and -independent pathways contribute to cAMP-mediated mitogenesis. Direct elevation of PI3K activity through microinjection of an antibody that stimulates PI3K activity or stable expression of membrane-localized p110 was sufficient to confer hormone-independent DNA synthesis when accompanied by elevations in p70s6k activity. These findings indicate that multiple pathways contribute to cAMP-stimulated mitogenesis, only some of which are PKA dependent. Furthermore, they demonstrate that the ability of cAMP to stimulate both p70s6k- and PI3K-dependent pathways is an important facet of cAMP-regulated cell cycle progression.

RhoB alteration is necessary for apoptotic and antineoplastic responses to farnesyltransferase inhibitors.

Farnesyltransferase inhibitors (FTIs) are in clinical trials, but how they selectively inhibit malignant cell growth remains uncertain. One important player in this process appears to be RhoB, an endosomal Rho protein that regulates receptor trafficking. FTI treatment elicits a gain of the geranylgeranylated RhoB isoform (RhoB-GG) that occurs due to modification of RhoB by geranylgeranyltransferase I in drug-treated cells. Notably, this event is sufficient to mediate antineoplastic effects in murine models and human carcinoma cells. To further assess this gain-of-function mechanism and determine whether RhoB-GG has a necessary role in drug action, we examined the FTI response of murine fibroblasts that cannot express RhoB-GG due to homozygous deletion of the rhoB gene. Nullizygous (-/-) cells were susceptible to cotransformation by adenovirus E1A plus activated H-Ras but defective in their FTI response, despite complete inhibition of H-Ras prenylation. Actin cytoskeletal and phenotypic events were disrupted in -/- cells, implicating RhoB-GG in these effects. Interestingly, -/- cells were resistant to FTI-induced growth inhibition under anchorage-dependent but not anchorage-independent conditions, indicating that, while RhoB-GG is sufficient, it is not necessary for growth inhibition under all conditions. In contrast, -/- cells were resistant to FTI-induced apoptosis in vitro and in vivo. Significantly, the apoptotic defect of -/- cells compromised the antitumor efficacy of FTI in xenograft assays. This study offers genetic proof of the hypothesis that RhoB-GG is a crucial mediator of the antineoplastic effects of FTIs.

Farnesyltransferase inhibition causes morphological reversion of ras-transformed cells by a complex mechanism that involves regulation of the actin cytoskeleton.

A potent and specific small molecule inhibitor of farnesyl-protein transferase, L-739,749, caused rapid morphological reversion and growth inhibition of ras-transformed fibroblasts (Rat1/ras cells). Morphological reversion occurred within 18 h of L-739,749 addition. The reverted phenotype was stable for several days in the absence of inhibitor before the transformed phenotype reappeared. Cell enlargement and actin stress fiber formation accompanied treatment of both Rat1/ras and normal Rat1 cells. Significantly, inhibition of Ras processing did not correlate with the initiation or maintenance of the reverted phenotype. While a single treatment with L-739,749 was sufficient to morphologically revert Rat1/ras cells, repetitive inhibitor treatment was required to significantly reduce cell growth rate. Thus, the effects of L-739,749 on transformed cell morphology and cytoskeletal actin organization could be separated from effects on cell growth, depending on whether exposure to a farnesyl-protein transferase inhibitor was transient or repetitive. In contrast, L-739,749 had no effect on the growth, morphology, or actin organization of v-raf-transformed cells. Taken together, the results suggest that the mechanism of morphological reversion is complex and may involve farnesylated proteins that control the organization of cytoskeletal actin.