Increased susceptibility to streptozotocin-induced beta-cell apoptosis and delayed autoimmune diabetes in alkylpurine-DNA-N-glycosylase-deficient mice

Type I diabetes is thought to occur as a result of the loss of insulin-producing pancreatic beta cells by an environmentally triggered autoimmune reaction. In rodent models of diabetes, streptozotocin (STZ), a genotoxic methylating agent that is targeted to the beta cells, is used to trigger the initial cell death. High single doses of STZ cause extensive beta -cell necrosis, while multiple low doses induce limited apoptosis, which elicits an autoimmune reaction that eliminates the remaining cells. We now show that in mice lacking the DNA repair enzyme alkylpurine-DNA-N-glycosylase (APNG), beta -cell necrosis was markedly attenuated after a single dose of STZ. This is most probably due to the reduction in the frequency of base excision repair-induced strand breaks and the consequent activation of poly(ADP-ribose) polymerase (PARP), which results in catastrophic ATP depletion and cell necrosis. Indeed, PARP activity was not induced in A-PNG(-/-) islet cells following treatment with STZ in vitro. However, 48 h after STZ treatment, there was a peak of apoptosis in the beta cells of APNG(-/-) mice. Apoptosis was not observed in PARP-inhibited APNG(+/+) mice, suggesting that apoptotic pathways are activated in the absence of significant numbers of DNA strand breaks. Interestingly, STZ-treated APNG(-/-) mice succumbed to diabetes 8 months after treatment, in contrast to previous work with PARP inhibitors, where a high incidence of beta -cell tumors was observed. In the multiple-low-dose model, STZ induced diabetes in both APNG(-/-) and APNG(-/-) mice; however, the initial peak of apoptosis was 2.5-fold greater in the APNG(-/-) mice. We conclude that APNG substrates are diabetogenic but by different mechanisms according to the status of APNG activity.

Primary Bartholin gland carcinoma: a report of seven cases

This study reviews our experience with 7 patients with primary Bartholin gland cancer (BGC) treated at the Queensland Gynaecological Cancer Centre (QCGC) and compares this with previously published data. A retrospective clinicopathologic review of all patients with primary BGC treated at QCGC from 1988 to 2000 was performed. Of the 7 patients treated, all underwent primary surgery and 5 of the 7 patients received radiotherapy postoperatively. All patients presented with a local swelling or a lump. Two had associated discharge and 2 had associated pain. Of the 7 patients, 2, 3 and 2 respectively were classified as having Stage IB, II or III disease. Five of the 7 patients had squamous cell carcinoma (SCC), one had adenoid-cystic carcinoma and 1 had a small-cell neuroendocrine cancer of the Bartholin gland. None of the patients with SCC developed recurrent disease. The patient with adenoid-cystic carcinoma experienced local recurrences at 4 years and again at 5 years and 3 months. Nine years after primary treatment she was diagnosed with pulmonary metastases. The patient with small-cell neuroendocrine cancer of the Bartholin gland was considered tumour-free after operation. Thorough imaging, including a CT scan of her chest, abdomen and pelvis showed no evidence of disease. She died 1 year and three months after diagnosis from disseminated pulmonary disease. We present the first report, of small cell neuroendocrine cancer of the Bartholin gland. Therapeutic principles in the management of vulval cancer at other sites appear to be appropriate for management of BGC.

Kinase-dependent and kinase-independent functions of EphA4 receptors in major axon tract formation in vivo

The EphA4 receptor tyrosine kinase regulates the formation of the corticospinal tract (CST), a pathway controlling voluntary movements, and of the anterior commissure (AC), connecting the neocortical temporal robes. To study EphA4 kinase signaling in these processes, we generated mice expressing mutant EphA4 receptors either lacking kinase activity or with severely downregulated kinase activity. We demonstrate that EphA4 is required for CST formation as a receptor for which it requires an active kinase domain. In contrast, the formation of the AC is rescued by kinase-dead EphA4, suggesting that in this structure EphA4 acts as a ligand for which its kinase activity is not required. Unexpectedly, the cytoplasmic sterile-alpha motif (SAM) domain is not required for EphA4 functions. Our findings establish both kinase-dependent and kinase-independent functions of EphA4 in the formation of major axon tracts.

Characterization of the EphA1 receptor tyrosine kinase: Expression in epithelial tissues

The Eph family (of receptor tyrosine kinases plays a crucial role during development and is implicated in oncogenesis. Using a partial cDNA clone of an Eph-related kinase (Esk) we isolated the complete coding region of a gene which we show to be murine EphA1 by both structural and functional criteria. The chromosomal localization is shown to be syntenic to hEphA1 and the genomic organization also shows distinct features found in the hEphA1 gene. Functionally, in keeping with findings for the human homologue, both soluble recombinant and native mEphA1 show preferential binding to ephrin A1. However, we also observed significant binding to other A-type ligands as has been observed for other Eph receptors. We analysed the expression of mEphA1 mRNA by in situ hybridization on tissue sections. mEphA1 was expressed in epithelial elements of skin, adult thymus, kidney and adrenal cortex. Taken together with previous Northern blotting data these results suggest that mEphA1 is expressed widely in differentiated epithelial cells.

Signals from Eph and ephrin proteins: a developmental tool kit

Interactions between Eph receptors and their ligands the ephrin proteins are critically important in many key developmental processes. Emerging evidence also supports a role for these molecules in postembryonic tissues, particularly in pathological processes, including tissue injury and tumor metastasis. We review the signaling mechanisms that allow the 14 Eph and nine ephrin proteins to deliver intracellular signals that regulate cell shape and movement. What emerges is that the initiation of these signals is critically dependent on which Eph and ephrin proteins are expressed, the level of their expression, and, in some cases, which splice variants are expressed. Diversity at the level of initial interaction and in the downstream signaling processes regulated by Eph-ephrin signaling provides a subtle, versatile system of regulation of intercellular adhesion, cell shape, and cell motility.

The progesterone receptor exon 4 Va1660Leu G/T polymorphism and risk of breast cancer in Australian women

An Alu insertion polymorphism of the progesterone receptor (PR) was reported recently to be associated with a reduced risk of breast cancer, with risks of 0.8- and 0.3-fold associated with the heterozygote and homozygote genotypes, respectively. This intronic variant is considered to be in linkage disequilibrium with an exon 4 hinge region G to T Val660Leu polymorphism. We investigated whether the exon 4 PR polymorphism was associated with breast cancer in Australian women, using a population-based study of 1452 cases and 793 controls, half of whom were

The BRCA2 372 HH genotype is associated with risk of breast cancer in Australian women under age 60 years

The BRCA2 N372H nonconservative amino acid substitution polymorphism appears to affect fetal survival in a sex-dependent manner, and the HH genotype was found to be associated with a 1.3-fold risk of breast cancer from pooling five case-control studies of Northern European women. We investigated whether the BR 2 N372H polymorphism was associated with breast cancer in Australian women using a population-based case-control design. The BRCA2 372 genotype was determined in 1397 cases under the age of 60 years at diagnosis of a first primary breast cancer and in 775 population-sampled controls frequency matched for age. Case-control analyses and comparisons of genotype distributions were conducted using logistic regression. All of the statistical tests were two-tailed. The HH genotype was independent of age and family history of breast cancer within cases and controls, and was more common in cases (9.2% versus 6.5%). It was associated with an increased risk of breast cancer, 1.47-fold unadjusted (95% confidence interval, 1.05-2.07; P = 0.02), and 1.42-fold (95% confidence interval, 1.00-2.02; P = 0.05) after adjusting for measured risk factors. This effect was still evident after excluding women with any non-Caucasian ancestry or the 33 cases known to have inherited a mutation in BRCA1 or BRCA2, and would explain similar to3% of breast cancer. The BRCA2 N372H polymorphism appears to be associated with a modest recessively inherited risk of breast cancer in Australian women. This result is consistent with the findings for Northern European women.

The pathogenesis of oral lichen planus

Both antigen-specific and non-specific mechanisms may be involved in the pathogenesis of oral lichen planus (OLP). Antigen-specific mechanisms in OLP include antigen presentation by basal keratinocytes and antigen-specific keratinocyte killing by CD8(+) cytotoxic T-cells. Non-specific mechanisms include mast cell degranulation and matrix metalloproteinase (MMP) activation in OLP lesions. These mechanisms may combine to cause T-cell accumulation in the superficial lamina propria, basement membrane disruption, intra-epithelial T-cell migration, and keratinocyte apoptosis in OLP. OLP chronicity may be due, in part, to deficient antigen-specific TGF-beta1-mediated immunosuppression. The normal oral mucosa may be an immune privileged site (similar to the eye, testis, and placenta), and breakdown of immune privilege could result in OLP and possibly other autoimmune oral mucosal diseases. Recent findings in mucocutaneous graft-versus-host disease, a clinical and histological correlate of lichen planus, suggest the involvement of TNF-alpha, CD40, Fas, MMPs, and mast cell degranulation in disease pathogenesis. Potential roles for oral Langerhans cells and the regional lymphatics in OLP lesion formation and chronicity are discussed. Carcinogenesis in OLP may be regulated by the integrated signal from various tumor inhibitors (TGF-beta1, TNF-alpha, IFN-gamma, IL-12) and promoters (MIF, MMP-9). We present our recent data implicating antigen-specific and non-specific mechanisms in the pathogenesis of OLP and propose a unifying hypothesis suggesting that both may be involved in lesion development. The initial event in OLP lesion formation and the factors that determine OLP susceptibility are unknown.

Alterations in gene expression and activity during squamous cell carcinoma development

This study focuses on characterizing the genetic and biological alterations associated with squamous cell carcinoma development. Normal human epidermal keratinocytes (HEKs), cells isolated from a preneoplastic lesion (IEC-1), and two neoplastic cell lines, SCC-25 and COLD-16, were grown as raft cultures, and their gene expression profiles were screened using cDNA arrays. Our data indicated that the expression levels of at least 37 genes were significantly (P less than or equal to 0.05; 1.9% of genes screened) altered in neoplastic cells compared with normal cells. Of these genes, 10 genes were up-regulated and 27 genes were down-regulated in the neoplastic cells. In addition, 51% of the genes altered in the neoplastic cells were already altered in the preneoplastic IEC-1 cells. Immunohistochemical staining of patient tumors was used to verify the cDNA array analysis. Our analysis indicated that alterations in genes associated with extracellular matrix production and apoptosis are disrupted in preneoplastic cells, whereas later stages of neoplasia are associated with alterations in gene expression for genes involved in DNA repair or epidermal growth factor (EGF) receptor/mitogen-activated protein kinase kinase (MAPKK)/MAPK/activator protein-1 (AP-1) signaling. Subsequent functional analysis of the alterations in expression of the EGF receptor/MAPKK/MAPK/AP-1 genes suggested they did not contribute to the neoplastic phenotype.

Nuclear PTEN expression and clinicopathologic features in a population-based series of primary cutaneous melanoma

Germline mutations of the PTEN tumor-suppressor gene, on 10q23, cause Cowden syndrome, an inherited hamartoma syndrome with a high risk of breast, thyroid and endometrial carcinomas and, some suggest, melanoma. To date, most studies which strongly implicate PTEN in the etiology of sporadic melanomas have depended on cell lines, short-term tumor cultures and noncultured metastatic melanomas. The only study which reports PTEN protein expression in melanoma focuses on cytoplasmic expression, mainly in metastatic samples. To determine how PTEN contributes to the etiology or the progression of primary cutaneous melanoma, we examined cytoplasmic and nuclear PTEN expression against clinical and pathologic features in a population-based sample of 150 individuals with incident primary cutaneous melanoma. Among 92 evaluable samples, 30 had no or decreased cytoplasmic PTEN protein expression and the remaining 62 had normal PTEN expression. In contrast, 84 tumors had no or decreased nuclear expression and 8 had normal nuclear PTEN expression. None of the clinical features studied, such as Clark's level and Breslow thickness or sun exposure, were associated with cytoplasmic PTEN expressional levels. An association with loss of nuclear PTEN expression was indicated for anatomical site (p = 0.06) and mitotic index (p = 0.02). There was also an association for melanomas to either not express nuclear PTEN or to express p53 alone, rather than both simultaneously (p = 0.02). In contrast with metastatic melanoma, where we have shown previously that almost two-thirds of tumors have some PTEN inactivation, only one-third of primary melanomas had PTEN silencing. This suggests that PTEN inactivation is a late event likely related to melanoma progression rather than initiation. Taken together with our previous observations in thyroid and islet cell tumors, our data suggest that nuclear-cytoplasmic partitioning of PTEN might also play a role in melanoma progression. (C) 2002 Wiley-Liss, Inc.

Laminin 10/11: an alternative adhesive ligand for epidermal keratinocytes with a functional role in promoting proliferation and migration

We have investigated the expression and function of the isoforms of laminin bearing the alpha(5) chain, i.e. laminin-10/11 in neonatal and adult human skin. By immunostaining human skin derived from a variety of anatomic sites, we found that the laminin-alpha(5) chain is expressed abundantly in the basement membrane underlying the interfollicular epidermis and the blood vessels in the dermis. Interestingly, while the expression level of the well-studied laminin-5 isoform did not change significantly with age, laminin-10/11 (a5 chain) appeared to decrease in the basement membrane underlying the epidermis, in adult skin. In contrast, the levels of laminin-10/11 in the basement membrane underlying blood vessels remained unchanged in neonatal vs. adult skin. Importantly, in vitro cell adhesion assays demonstrated that laminin-10/11 is a potent adhesive substrate for both neonatal and adult keratinocytes and that this adhesion is mediated by the alpha(3)beta(1), and alpha(6)beta(4) integrins. Adhesion assays performed with fractionated basal keratinocytes showed that stem cells, transit amplifying cells and early differentiating cells all adhere to purified laminin-10/11 via these receptors. Further, laminin-10/11 provided a proliferative signal for neonatal foreskin keratinocytes, adult breast skin keratinocytes, and even a human papillomavirus type-18 transformed tumorigenic keratinocyte cell line in vitro. Finally, laminin-10/11 was shown to stimulate keratinocyte migration in an in vitro wound healing assay. These results provide strong evidence for a functional role for laminin-10/11 in epidermal proliferation during homeostasis, wound healing and neoplasia.

The impact of nutrition support on body composition in cancer outpatients receiving radiotherapy

This study investigated the change in body composition in 36 cancer outpatients receiving radiotherapy to the head and neck area (mean age: 63 ± 15 years) randomised to receive either nutrition intervention (NI; n=15) or usual care (UC; n=21). Body weight and composition were measured at the commencement of radiotherapy and 3 months later. The UC group lost significantly more weight; mean decrease = 4.3 kg, than the NI group: mean decrease = 1.1 kg (t(30)=-2.5, p=0.019). Fat-free mass loss was significantly higher in the UC group with a mean loss of 2.2 kg versus 0.3 kg in the NI group (t(30)=- 2.3, p=0.029). Body composition as measured by foot-to-foot bioelectrical impedance analysis provides more information than weight alone and can allow for tailoring of NI.