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.

Immunohistochemistry versus microsatellite instability testing in phenotyping colorectal tumors

Purpose: To compare microsatellite instability (MSI) testing with immunohistochemical (IHC) detection of hMLH1 and hMSH2 in colorectal cancer. Patients and Methods: Colorectal cancers from 1, 144 patients were assessed for DNA mismatch repair deficiency by two methods: MSI testing and IHC detection of hMLH1 and hMSH2 gene products. High-frequency MSI (MSI-H) was defined as more than 30% instability of at least five markers; low-level MSI (MSI-L) was defined as 1% to 29% of loci unstable. Results: Of 1, 144 tumors tested, 818 showed intact expression of hMLH1 and hMSH2. Of these, 680 were microsatellite stable (MSS), 27 were MSI-H, and 111 were MSI-L. In all, 228 tumors showed absence of hMLH1 expression and 98 showed absence of hMSH2 expression: all were MSI-H. Conclusion: IHC in colorectal tumors for protein products hMLH1 and hMSH2 provides a rapid, cost-effective, sensitive (92.3%), and extremely specific (100%) method for screening for DNA mismatch repair defects. The predictive value of normal IHC for an MSS/MSI-L phenotype was 96.7%, and the predictive value of abnormal IHC was 100% for an MSI-H phenotype. Testing strategies must take into account acceptability of missing some cases of MSI-H tumors if only IHC is performed. (C) 2002 by American Society of Clinical Oncology.

Mutations in exon 3 of the beta-catenin gene are rare in melanoma cell lines

Mutations in exon 3 of the CTNNB1 gene encoding beta-catenin have been reported in colorectal cancer cell lines and tumours. Although one study reported mutations or deletions affecting beta-catenin in 20% of melanoma cell lines, subsequent reports detected a much lower frequency of aberrations in uncultured melanomas. To determine whether this difference in mutation frequency reflected an in vitro culturing artefact, exon 3 of CTNNB1 was screened in a panel of 62 melanoma cell lines. In addition, reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect intragenic deletions affecting exon 3. One out of 62 (1.6%) cell lines was found to carry a mutation, indicating that aberration of the Wnt-l/wingless pathway through activation of beta-catenin is a rare event, even in melanoma cell lines. (C) 2002 Lippincott Williams Wilkins.

Hypertension in Pagothenia borchgrevinki caused by X-cell disease

Approximately 15% of a population of the cryopelagic nototheniid fish Pagothenia borchgrevinki, found constantly swimming immediately beneath the annual fast ice, in McMudro Sound. Ross Sea, Antarctica, was affected by X-cell gill disease. This disease affected blood flow through the gill lamellae, and this in turn affected oxygen uptake. Exercise caused increases in heart rate and ventral aortic blood pressure. Heart rate increased from 15.1 +/- 1.55 to 23.1 +/- 0.93 beats min(-1) in healthy fish, with a similar increase from 15.1 +/- 1.55 to 23.1 +/- 0.93 beats min(-1) in healthy fish, with a similar increase (to 24.6 +/- 0.26 beats min(-1)) in X-cell-affected animals. In healthy fish, pressures rose with exercise (from 2.72 +/- 0.11 to 3.75 +/- 0.19 kPa) and then rapidly returned to resting levels during recovery. In X-cell fish pressures rose during exercise, but then continued to rise, to reach a high of 4.18 +/- 0.13 kPa, close to the predicted maximum pressure able to be generated by these hearts. Recovery was rapid in healthy fish, but was prolonged in diseased animals. As they are constantly swimming, there is the potential that X-cell-affected fish suffer from chronic hypertension. (C) 2003 The Fisheries Society of the British Isles.

Antitumor activity of 3-ingenyl angelate: Plasma membrane and mitochondrial disruption and necrotic cell death

Options for skin cancer treatment currently include surgery, radiotherapy, topical chemotherapy, cryosurgery, curettage, and electrodes-sication. Although effective, surgery is costly and unsuitable for certain patients. Radiotherapy can leave a poor cosmetic effect, and current chemotherapy is limited by low cure rates and extended treatment schedules. Here, we describe the preclinical activity of a novel topical chemotherapeutic agent for the treatment of skin cancer, 3-ingenyl angelate (PEP005), a hydrophobic diterpene ester isolated from the plant Euphorbia peplus. Three daily topical applications of 42 nmol (18 mug) of PEP005 cured a series of s.c. mouse tumors (B16 melanoma, LK2 UV-induced squamous cell carcinoma, and Lewis lung carcinoma; it = >14 tumors/group) and human tumors (DO4 melanoma, HeLa cervical carcinoma, and PC3 and DU145 prostate carcinoma; it = >4 tumors/group) previously established (5-10 mm(3)) on C57BL/6 or Fox1(nu) mice. The treatment produced a mild, short-term erythema and eschar formation but, ultimately, resulted in excellent skin cosmesis. The LD90 for PEP005 for a panel of tumor cell lines was 180-220 muM. Electron microscopy showed that treatment with PEP005 both ill vitro (230 tot) and ill vivo (42 nmol) rapidly caused swelling of mitochondria and cell death by primary necrosis. Cr-51 release, uptake of propidium iodide, and staining with the mitochondria dye JC1, revealed that PEP005 (230 muM) treatment of tumor cells ill vitro resulted in a rapid plasma membrane perturbation and loss of mitochondrial membrane potential. PEP005 thus emerges as a new topical anti-skin cancer agent that has a novel mode of action involving plasma membrane and mitochondrial disruption and primary necrosis, ultimately resulting in an excellent cosmetic outcome.

Induction of Therapeutic T-Cell Responses to Subdominant Tumor-associated Viral Oncogene after Immunization with Replication-incompetent Polyepitope Adenovirus Vaccine

The EBV-encoded latent membrane proteins (LMP1 and LMP2), which are expressed in various EBV-associated malignancies have been proposed as a potential target for CTL-based therapy. However, the precursor frequency for LMP-specific CTL is generally low, and immunotherapy based on these antigens is often compromised by the poor immunogenicity and potential threat from their oncogenic potential. Here we have developed a replication-incompetent adenoviral vaccine that encodes multiple HLA class I-restricted CTL epitopes from LMP1 and LMP2 as a polyepitope. Immunization with this polyepitope vaccine consistently generated strong LMP-specific CTL responses in HLA A2/K-b mice, which can be readily detected by both ex vivo and in vivo T-cell assays. Furthermore, a human CTL response to LMP antigens can be rapidly expanded after stimulation with this recombinant polyepitope vector. These expanded T cells displayed strong lysis of autologous target cells sensitized with LMP1 and/or LMP2 CTL epitopes. More importantly, this adenoviral vaccine was also successfully used to reverse the outgrowth of LMP1-expressing tumors in HLA A2/K-b mice. These studies demonstrate that a replication-incompetent adenovirus polyepitope vaccine is an excellent tool for the induction of a protective CTL response directed toward multiple LMP CTL epitopes restricted through common HLA class I alleles prevalent in different ethnic groups where EBV-associated malignancies are endemic.

Recombinant Kunjin virus replicon vaccines induce protective T-cell immunity against human papillomavirus 16 E7-expressing tumour

The persistence of the E7 oncoprotein in transformed cells in human papillomavirus (HPV)-associated cervical cancer provides a tumour-specific antigen to which immunotherapeutic strategies may be directed. Self-replicating RNA (replicon) vaccine vectors derived from the flavivirus Kunjin (KUN) have recently been reported to induce T-cell immunity. Here, we report that inclusion of a CTL epitope of HPV16 E7 protein into a polyepitope encoded by a KUN vector induced E7-directed T-cell responses and protected mice against challenge with an E7-expressing epithelial tumour. We found replicon RNA packaged into virus-like particles to be more effective than naked replicon RNA or plasmid DNA constructed to allow replicon RNA transcription in vivo. Protective immunity was induced although the E7 CTL epitope was subdominant in the context of other CTL epitopes in the polyepitope. The results demonstrate the efficacy of the KUN replicon vector system for inducing protective immunity directed towards a virally encoded human tumour-specific antigen, and for inducing multi-epitopic CTL responses. (C) 2004 Elsevier Inc. All rights reserved.

Endothelin axis expression is markedly different in the two main subtypes of renal cell carcinoma

BACKGROUND. The endothelin axis has been implicated in cancer growth, angiogenesis, and metastasis, but to the authors' knowledge the expression of endothelin genes has not been defined in renal cell carcinoma (RCC). METHODS. Tissue specimens were harvested from both normal and tumor-affected regions at the time of radical nephrectomy from 35 patients with RCC (22 with clear cell RCC [ccRCC] and 13 with papillary RCC [PRCC]). Real-time reverse transcriptase-polymerase chain reaction analysis determined the expression profile of the preproendothelins (PPET-1, PPET-2, and PPET-3), the endothelin receptors (ETA and ETB), and the endothelin-converting enzymes (ECE-1 and ECE-2). RESULTS. PPET-1 was found to be up-regulated in ccRCC tumor specimens and down-regulated in PRCC tumor specimens. ETA was significantly down-regulated in PRCC tumor specimens. ECE-1 was expressed in all tissue specimens at comparable levels, with moderate but significant elevation in normal tissue specimens associated with PRCC. Of the other genes, PPET-2 and ETB were expressed in all tissue specimens and no differences were observed between tumor subtypes or tumor-affected and normal tissue specimens, whereas PPET-3 and ECE-2 were present in all tissue specimens but were barely detectable. CONCLUSIONS. The endothelin axis was expressed differently in the two main subtypes of RCC and appeared to match macroscopic features commonly observed in these tumors (i.e., high expression of PPET-I in hypervascular ccRCC contrasted against low PPET-1 and ETA expression in hypovascular PRCC). The presence of ECE-1 mRNA in these tissue specimens suggested that active endothelin ligands were present, indicating endothelin axis activity was elevated in ccRCC compared with normal kidney, but impaired in PRCC. The current study provided further evidence that it is not appropriate to consider ccRCC and PRCC indiscriminately in regard to treatment. (C) 2004 American Cancer Society.

Differential proliferative response of NKT cell subpopulations to in vitro stimulation in presence of different cytokines

Human Valpha24(+)Vbeta11(+) NKT (NKT) cells have immune regulatory activities associated with rejection of tumors, infections and control of autoimmune diseases. They can be stimulated to proliferate using alpha-galactosylceramide (KRN7000) and have the potential for therapeutic manipulation. Subpopulations of NKT cells (CD4(+)CD8(-), CD4(-)D8(+) and CD4(-)CD8(-)) have functionally distinctive Th1/Th2 cytokine profiles and their relative numbers following stimulation may influence the Th1/Th2 balance, which may result in or prevent disease. We aimed to determine the effect of different cytokines in culture during stimulation of NKT cells on the relative proportions of NKT cell subpopulations. Our results show that all NKT cell subpopulations expanded following stimulation with KRN7000 and IL-2, IL-7, IL-1 2 or IL-15. Expansion capacity differed between subpopulations, resulting in different relative proportions of CD4(+) and CD4(-) NKT cell subpopulations, and this was influenced by the cytokine used for stimulation. A Th1-biased environment was observed after stimulation of NKT cells. NKT cells expanded under all conditions evaluated demonstrated significant cytotoxicity against U937 tumor cells. In view of the potential for NKT cell subsets to alter the balance of Th1 and Th2 environment, these data provide insights into the effects of NKT cell manipulation for possible therapeutic applications in different disease settings.

Brca1 inactivation induces p27Kip1-dependent cell cycle arrest and delayed development in the mouse mammary gland

One common characteristic of breast cancers arising in carriers of the predisposition gene BRCA1 is a loss of expression of the CDK inhibitor p27(Kip1) (p27), suggesting that p27 interacts epistatically with BRCA1. To investigate this relationship, we examined expression of p27 in mice expressing a dominant negative allele of Brca1 (MMTV-trBr) in the mammary gland. While these mice rarely develop tumors, they showed a 50% increase in p27 protein and a delay in mammary gland development associated with reduced proliferation. In contrast, on a p27 heterozygote background, MMTV-trBrca1 mice showed an increase in S phase cells, and normal mammary development. p27 was the only protein in the cyclin cyclin-dependent kinase network to show altered expression, suggesting that it may be a central mediator of cell cycle arrest in response to loss of function of BRCA1. Furthermore, in human mammary epithelial MCF7 cells expressing BRCA1-specific RNAi and in the BRCA1-deficient human tumor cell line HCC1937, p27 is elevated at the mRNA level compared to cells expressing wild-type BRCA1. We hypothesize that disruption of BRCA1 induces an increase in p27 that inhibits proliferation. Accordingly, reduction in p27 expression leads to enhancement of cellular proliferation in the absence of BRCA1.