Molecular mechanism of PUVA-induced apoptosis in mouse epidermal cells

A combination of psoralens and ultraviolet-A radiation referred to as PUVA, is widely used in the treatment of psoriasis. PUVA therapy is highly effective in killing hyperproliferative cells, but its mechanism of action has not been fully elucidated. Psoralen binds to DNA, and upon photoactivation by UVA, it forms monofunctional adducts and interstrand cross-links. PUVA treatment has been shown to be mutagenic and to produce tumors in animals. In addition, epidemiological studies have reported a 10 to 15 percent increased risk of developing squamous cell carcinoma in individuals treated chronically with PUVA. However, it remains a treatment for skin disorders such as psoriasis because its benefits outweigh its risks. The widespread use of PUVA therapy and its associated cancer risk requires us to understand the molecular mechanisms by which PUVA induces cell death. Immortalized JB6 mouse epidermal cells, p53−/− mice, and Fas Ligand−/− (gld) mice were used to investigate the molecular mechanism by which PUVA kills cells. Treatment of JB6 cells with 10 μg/ml 8-methoxypsoralen followed by irradiation with 20 kJ/m2 UVA resulted in cell death. The cells exhibited morphological and biochemical characteristics of apoptosis such as chromatin condensation, DNA ladder formation, and TUNEL-positivity. PUVA treatment stabilized and phosphorylated p53 leading to its activation, as measured by nuclear localization and induction of p21Waf/Cip1, a transcriptional target of p53. Subsequent in vivo studies revealed that there was statistically significantly less apoptosis in p53 −/− mice than in p53+/+ mice at 72 hours after PUVA. In addition, immunohistochemical analysis revealed more Fas and FasL expression in p53+/+ mice than in p53−/− mice, suggesting that p53 is required to transcriptionally activate Fas, which in turn causes the cells to undergo apoptosis. Studies with gld mice confirmed a role for Fas/FasL interactions in PUVA-induced apoptosis. There was statistically significantly less apoptosis in gld mice compared with wild-type mice 24, 48, and 72 hours after PUVA. These results demonstrate that PUVA-induced apoptosis in mouse epidermal cells requires p53 and Fas/FasL interactions. These findings may be important for designing effective treatments for diseases such as psoriasis without increasing the patient's risk for skin cancer. ^

The transcriptional repressor Delta EF1 controls resistance to the EGFR inhibitor erlotinib in human HNSCC lines

Epidermal Growth Factor Receptor (EGFR) overexpression occurs in about 90% of Head and Neck Squamous Cell Carcinoma (HNSCC) cases. Aberrant EGFR signaling has been implicated in the malignant features of HNSCC. Thus, EGFR appears to be a logical therapeutic target with increased tumor specificity for the treatment of HNSCC. Erlotinib, a small molecule tyrosine kinase inhibitor, specifically inhibits aberrant EGFR signaling in HNSCC. Only a minority of HNSCC patients were able to derive a substantial clinical benefit from erlotinib. ^ This dissertation identifies Epithelial to Mesenchymal Transition (EMT) as the biological marker that distinguishes EGFR-dependent (erlotinib-sensitive) tumors from the EGFR-independent (erlotinib-resistant) tumors. This will allow us to prospectively identify the patients who are most likely to benefit from EGFR-directed therapy. More importantly, our data identifies the transcriptional repressor DeltaEF1 as the mesenchymal marker that controls EMT phenotype and resistance to erlotinib in human HNSCC lines. si-RNA mediated knockdown of DeltaEF1 in the erlotinib-resistant lines resulted in reversal of the mesenchymal phenotype to an epithelial phenotype and significant increase in sensitivity to erlotinib. ^ DeltaEF1 represses the expression of the epithelial markers by recruiting HDACs to chromatin. This observation allows us to translate our findings into clinical application. To test whether the transcriptional repression by DeltaEF1 underlines the mechanism responsible for erlotinib resistance, erlotinib-resistant lines were treated with an HDAC inhibitor (SAHA) followed by erlotinib. This resulted in a synergistic effect and substantial increase in sensitivity to erlotinib in the resistant cell lines. Thus, combining an HDAC inhibitor with erlotinib represents a novel promising pharmacologic strategy for reversing resistance to erlotinib in HNSCC patients. ^

Thrombospondin-2: A potent endogenous inhibitor of tumor growth and angiogenesis

Recent evidence suggests a potential role for thrombospondin-2 (TSP-2), a matricellular glycoprotein, in the regulation of primary angiogenesis. To directly examine the biological effect of TSP-2 expression on tumor growth and angiogenesis, human A431 squamous cell carcinoma cells, which do not express TSP-2, were stably transfected with a murine TSP-2 expression vector or with vector alone. A431 cells expressing TSP-2 did not show an altered growth rate, colony-forming ability, or susceptibility to induction of apoptosis in vitro. However, injection of TSP-2-transfected clones into the dermis of nude mice resulted in pronounced inhibition of tumor growth that was significantly stronger than the inhibition observed in A431 clones stably transfected with a thrombospondin-1 (TSP-1) expression vector, and combined overexpression of TSP-1 and TSP-2 completely prevented tumor formation. Extensive areas of necrosis were observed in TSP-2-expressing tumors, and both the density and the size of tumor vessels were significantly reduced, although tumor cell expression of the major tumor angiogenesis factor, vascular endothelial growth factor, was maintained at high levels. These findings establish TSP-2 as a potent endogenous inhibitor of tumor growth and angiogenesis.

Defects in transforming growth factor-β signaling cooperate with a Ras oncogene to cause rapid aneuploidy and malignant transformation of mouse keratinocytes

Genetic inactivation of the transforming growth factor-β (TGF-β) signaling pathway can accelerate tumor progression in the mouse epidermal model of multistage carcinogenesis. By using an in vitro model of keratinocyte transformation that parallels in vivo malignant conversion to squamous cell carcinoma, we show that v-rasHa transduced primary TGF-β1−/− keratinocytes and keratinocytes expressing a TGF-β type II dominant-negative receptor transgene have significantly higher frequencies of spontaneous transformation than control genotypes. Malignant transformation in the TGF-β1−/− keratinocytes is preceded by aneuploidy and accumulation of chromosomal aberrations. Similarly, transient inactivation of TGF-β signaling with a type II dominant-negative receptor adenovirus causes rapid changes in ploidy. Exogenous TGF-β1 can suppress aneuploidy, chromosome breaks, and malignant transformation of the TGF-β1−/− keratinocytes at concentrations that do not significantly arrest cell proliferation. These results point to genomic instability as a mechanism by which defects in TGF-β signaling could accelerate tumor progression in mouse multistage carcinogenesis.

The reactive site loop of the serpin SCCA1 is essential for cysteine proteinase inhibition

The high-molecular-weight serine proteinase inhibitors (serpins) are restricted, generally, to inhibiting proteinases of the serine mechanistic class. However, the viral serpin, cytokine response modifier A, and the human serpins, antichymotrypsin and squamous cell carcinoma antigen 1 (SCCA1), inhibit different members of the cysteine proteinase class. Although serpins employ a mobile reactive site loop (RSL) to bait and trap their target serine proteinases, the mechanism by which they inactivate cysteine proteinases is unknown. Our previous studies suggest that SCCA1 inhibits papain-like cysteine proteinases in a manner similar to that observed for serpin–serine proteinase interactions. However, we could not preclude the possibility of an inhibitory mechanism that did not require the serpin RSL. To test this possibility, we employed site-directed mutagenesis to alter the different residues within the RSL. Mutations to either the hinge or the variable region of the RSL abolished inhibitory activity. Moreover, RSL swaps between SCCA1 and the nearly identical serpin, SCCA2 (an inhibitor of chymotrypsin-like serine proteinases), reversed their target specificities. Thus, there were no unique motifs within the framework of SCCA1 that independently accounted for cysteine proteinase inhibitory activity. Collectively, these data suggested that the sequence and mobility of the RSL of SCCA1 are essential for cysteine proteinase inhibition and that serpins are likely to utilize a common RSL-dependent mechanism to inhibit both serine and cysteine proteinases.

Cytologically normal cells from neoplastic cervical samples display extensive structural abnormalities on IR spectroscopy: Implications for tumor biology

Fourier-transform IR (FT-IR) spectra of pelleted exfoliated cervical cells from patients with cervical cancer or dysplasia differ from those from normal women. To study the origin of these spectral changes, we obtained the FT-IR spectra of individual cervical cells from normal, dysplastic, and malignant cervical samples. Ninety five percent of normal superficial and intermediate cells displayed two distinct spectral patterns designated A and B, and 5% displayed an intermediate pattern, suggesting extensive structural heterogeneity among these cells. Parabasal and endocervical cells showed pattern B spectra. The spectra of malignant, dysplastic, and other abnormal cells also were characterized. Analysis of FT-IR spectra of over 2,000 individual cells from 10 normal females, 7 females with dysplasia, and 5 females with squamous cell carcinoma revealed that the spectra of normal-appearing intermediate and superficial cells of the cervix from women with either dysplasia or cancer differed from those of normal women. Chemometric and classical spectroscopic analysis showed a continuum of changes paralleling the transition from normalcy to malignancy. These findings suggest that (i) the structural changes underlying the spectroscopic changes are involved in or are a product of cervical carcinogenesis and (ii) the neoplastic process may be more extensive than currently recognized with morphological criteria. This approach may be useful for the structural study of neoplasia and also may be of help in the diagnosis or classification of cervical disorders.

p53 associates with and targets ΔNp63 into a protein degradation pathway

A human p53 homologue, p63 (p40/p51/p73L/CUSP) that maps to the chromosomal region 3q27–29 was found to produce a variety of transcripts that encode DNA-binding proteins with and without a trans-activation domain (TA- or ΔN-, respectively). The p63 gene locus was found to be amplified in squamous cell carcinoma, and overexpression of ΔNp63 (p40) led to increased growth of transformed cells in vitro and in vivo. Moreover, p63-null mice displayed abnormal epithelial development and germ-line human mutations were found to cause ectodermal dysplasia. We now demonstrate that certain p63 isotypes form complexes with p53. p53 mutations R175H or R248W abolish the association of p53 with p63, whereas V143A or R273H has no effect. Deletion studies suggest that the DNA-binding domains of both p53 and p63 mediate the association. Overexpression of wild type but not mutant (R175H) p53 results in the caspase-dependent degradation of certain ΔNp63 proteins (p40 and ΔNp63α). The association between p53 and ΔNp63 supports a previously unrecognized role for p53 in regulation of ΔNp63 stability. The ability of p53 to mediate ΔNp63 degradation may balance the capacity of ΔNp63 to accelerate tumorigenesis or to induce epithelial proliferation.

Cloning and sequencing of CATR1.3, a human gene associated with tumorigenic conversion.

The human squamous cell carcinoma cell line SCC83-01-82 (SCC) contains mutations in both the H-ras and p53 genes, but it exhibits a nontumorigenic phenotype in nude mice. This cell line can be converted into a cell line with a tumorigenic phenotype, SCC83-01-82CA (CA), by treatment with the mutagen methyl methanesulfonate (MMS). This indicates that additional genetic events leading to expression of a cooperating tumor susceptibility gene(s) may be required for tumorigenicity. To identify the cooperating gene(s), an expression cDNA library was made from tumorigenic Ca cells. The library DNA was transfected into nontumorigenic SCC cells and the transfected SCC cells were then injected into nude mice for the selection of a tumorigenic phenotype. Tumors developed in 3 of the 18 mice after injection. Several new cell lines were established from these transfected cell-induced tumors and designated as CATR cells. Tumor histology and karyotype analysis of these cells indicated that they were of human epithelial cell origin. All the CATR cells have the library vector sequence integrated in their genome. Cell line CATR1 expressed a single message from the integrated library representing a 1.3-kb cDNA insert that was absent from untransfected SCC cells or MMS-converted CA cells. This 1.3-kb cDNA insert was cloned by PCR amplification of reverse-transcribed CATR1 total RNA and was designated CATR1.3. The nucleotide sequence of CATR1.3 encodes a peptide of 79 amino acids, has a long 3' untranslated region, and represents an unknown gene product that was associated with the tumorigenic conversion due to the transfected expression library.

Diabetes mellitus e câncer de cabeça e pescoço

O diabetes mellitus (DM) está associado com alguns tipos de câncer. No entanto, estudos realizados sobre a associação entre DM e câncer de cabeça e pescoço (CCP) apresentaram resultados controversos. Na avaliação da associação entre DM e câncer, destaque deve ser dado à metformina, medicamento utilizado no DM tipo 2, que se mostra inversamente associado a alguns tumores. O objetivo deste estudo foi avaliar a associação entre DM e CCP, bem como o impacto do uso de metformina no risco de CCP. Este estudo caso-controle incluiu 1021 casos de CCP com confirmação histológica de carcinoma espino celular selecionados em cinco hospitais de grande porte no estado de São Paulo entre 2011 e 2014. Os 1063 controles foram recrutados nos mesmos hospitais, pareados por frequência com os casos por sexo e idade (em grupos de 5 anos). Para avaliar o risco de CCP associado ao DM, odds ratios (OR) e intervalos com 95 por cento de confiança (IC 95 por cento ) foram estimados por meio de regressão logística não condicional. Os participantes diabéticos tiveram associação inversa com o CCP (OR = 0,68; IC 95 por cento : 0,49-0,95), e a proteção foi maior entre diabéticos usuários metformina (OR = 0,54; IC 95 por cento : 0,29-0,99). Diabéticos usuários de metformina que eram fumantes (OR = 0,13; IC 95 por cento : 0,04-0,44), ou consumidores de álcool acima de 40 g/ dia (OR = 0,31; IC 95 por cento : 0,11-0,88) apresentaram proteção ainda maior com relação ao CCP, comparado aos não diabéticos. Em conclusão, os indivíduos diabéticos apresentaram risco inverso de CCP e o uso de metformina pode explicar, ao menos parcialmente, esta associação.

Características clínico-epidemiológicas dos doentes com neoplasia da cabeça e pescoço referenciados ao departamento de otorrinolaringologia

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Quando a hiperémia conjuntival constitui um desafio : a propósito de um caso clínico de tumor epidermóide da conjuntiva

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Estrogen metabolites for the diagnosis of schistosomiasis associated urinary bladder cancer

Letter to the Editor

An Epidemiologic analysis of the morphologic types of cancer of the esophagus /

"April 1991."

Interactive effects of MC1R and OCA2 on melanoma risk phenotypes

The relationships between MC1R gene variants and red hair, skin reflectance, degree of freckling and nevus count were investigated in 2331 adolescent twins, their sibs and parents in 645 twin families. Penetrance of each MC1R variant allele was consistent with an allelic model where effects were multiplicative for red hair but additive for skin reflectance. Of nine MC1R variant alleles assayed, four common alleles were strongly associated with red hair and fair skin (Asp84Glu, Arg151Cys, Arg160Trp and Asp294His), with a further three alleles having low penetrance (Val60Leu, Val92Met and Arg163Gln). These variants were separately combined for the purposes of this analysis and designated as strong 'R' (OR=63.3; 95% CI 31.9-139.6) and weak 'r ' (OR=5.1; 95% CI 2.5-11.3) red hair alleles. Red-haired individuals are predominantly seen in the R/R and R/r groups with 67.1 and 10.8%, respectively. To assess the interaction of the brown eye color gene OCA2 on the phenotypic effects of variant MC1R alleles we included eye color as a covariate, and also genotyped two OCA2 SNPs (Arg305Trp and Arg419Gln), which were confirmed as modifying eye color. MC1R genotype effects on constitutive skin color, freckling and mole count were modified by eye color, but not genotype for these two OCA2 SNPs. This is probably due to the association of these OCA2 SNPs with brown/green not blue eye color. Amongst individuals with a R/R genotype (but not R/r), those who also had brown eyes had a mole count twice that of those with blue eyes. This suggests that other OCA2 polymorphisms influence mole count and remain to be described.

Molecular introduction to head and neck cancer (HNSCC) carcinogenesis

Of all human cancers, HNSCC is the most distressing affecting pain, disfigurement, speech and the basic survival functions of breathing and swallowing. Mortality rates have not significantly changed in the last 40 years despite advances in radiotherapy and surgical treatment. Molecular markers are currently being identified that can determine prognosis preoperatively by routine tumour biopsy Leading to improved management of HNSCC patients. The approach could help decide which early stage patient should have adjuvant neck dissection and radiotherapy, and whether Later stage patients with operable lesions would benefit from resection and reconstructive surgery or adopt a conservative approach to patients with poor prognosis regardless of treatment. In the future, understanding these basic genetic changes in HNSCC would be important for the management of HNSCC. (C) 2004 The British Association of Plastic Surgeons. Published by Elsevier Ltd. All rights reserved.