Interaction of the hepatitis B virus X protein with the Crm1-dependent nuclear export pathway.

The leucine-rich nuclear export signal (NES) is used to shuttle large cellular proteins from the nucleus to the cytoplasm. The nuclear export receptor Crm1 is essential in this process by recognizing the NES motif. Here, we show that the oncogenic hepatitis B virus (HBV) X protein (HBx) contains a functional NES motif. We found that the predominant cytoplasmic localization of HBx is sensitive to the drug leptomycin B (LMB), which specifically inactivates Crm1. Mutations at the two conserved leucine residues to alanine at the NES motif (L98A,L100A) resulted in a nuclear redistribution of HBx. A recombinant HBx protein binds to Crm1 in vitro. In addition, ectopic expression of HBx sequesters Crm1 in the cytoplasm. Furthermore, HBx activates NFkappaB by inducing its nuclear translocation in a NES-dependent manner. Abnormal cytoplasmic sequestration of Crm1, accompanied by a nuclear localization of NFkappaB, was also observed in hepatocytes from HBV-positive liver samples with chronic active hepatitis. We suggest that Crm1 may play a role in HBx-mediated liver carcinogenesis.

Increased p53 mutation load in nontumorous human liver of wilson disease and hemochromatosis: oxyradical overload diseases.

Hemochromatosis and Wilson disease (WD), characterized by the excess hepatic deposition of iron and copper, respectively, produce oxidative stress and increase the risk of liver cancer. Because the frequency of p53 mutated alleles in nontumorous human tissue may be a biomarker of oxyradical damage and identify individuals at increased cancer risk, we have determined the frequency of p53 mutated alleles in nontumorous liver tissue from WD and hemochromatosis patients. When compared with the liver samples from normal controls, higher frequencies of G:C to T:A transversions at codon 249 (P < 0.001) and C:G to A:T transversions and C:G to T:A transitions at codon 250 (P < 0.001 and P < 0.005) were found in liver tissue from WD cases, and a higher frequency of G:C to T:A transversions at codon 249 (P < 0.05) also was found in liver tissue from hemochromatosis cases. Sixty percent of the WD and 28% of hemochromatosis cases also showed a higher expression of inducible nitric oxide synthase in the liver, which suggests nitric oxide as a source of increased oxidative stress. A high level of etheno-DNA adducts, formed from oxyradical-induced lipid peroxidation, in liver from WD and hemochromatosis patients has been reported previously. Therefore, we exposed a wild-type p53 TK-6 lymphoblastoid cell line to 4-hydroxynonenal, an unsaturated aldehyde involved in lipid peroxidation, and observed an increase in G to T transversions at p53 codon 249 (AGG to AGT). These results are consistent with the hypothesis that the generation of oxygen/nitrogen species and unsaturated aldehydes from iron and copper overload in hemochromatosis and WD causes mutations in the p53 tumor suppressor gene.

Gene-modified PA1-STK cells home to tumor sites in patients with malignant pleural mesothelioma.

BACKGROUND: Malignant mesothelioma is an uncommon but lethal cancer of increasing incidence, particularly among patients with a history of exposure to asbestos. Although numerous treatments have been employed, including chemotherapy, radiation therapy, surgical resection, and combinations of the above, no satisfactory treatment yet exists, and affected patients will die of this disease, usually within 12 months. Gene-based therapies constitute a new approach that offers hope of improved control of these tumors while being associated with less morbidity than conventional chemotherapeutic or surgical regimens. We demonstrated that PA1-STK cells home in vivo to mesothelioma deposits, a phenomenon that is required for optimal exertion of this therapeutic concept. METHODS: Gene-modified ovarian cancer cells expressing the thymidine-kinase gene (PA1-STK) were radiolabeled with 99Tc and infused into the pleural space of 4 patients with malignant pleural mesothelioma, then scanned to determine distribution of the cells. RESULTS: PA1-STK cells recognized and adhered preferentially to mesothelioma lining the chest wall. CONCLUSIONS: Cell-based "suicide gene" therapy utilizing the "bystander effect" with the gene-modified ovarian cancer cell line PA1-STK is feasible in human pleural mesothelioma. We have shown that this trafficking and homing of the therapeutic cells to the intrapleural tumor sites, a requirement for success with this novel therapeutic concept, is also valid in humans.

Increased p53 mutation load in noncancerous colon tissue from ulcerative colitis: a cancer-prone chronic inflammatory disease.

Ulcerative colitis (UC) is a chronic inflammatory disease that produces reactive oxygen and nitrogen species and increases the risk of colorectal cancer (CRC). The p53 tumor suppressor gene is frequently mutated in UC-associated dysplastic lesions and CRC. We are exploring the hypothesis that p53 mutations in the nontumorous colonic tissue in noncancerous UC cases indicate genetic damage from exposure to exogenous and endogenous carcinogens and may identify individuals at increased cancer risk. We are reporting, for the first time, the frequency of specific p53 mutated alleles in nontumorous colon tissue from donors either with or without UC by using a highly sensitive genotypic mutation assay. Higher p53 mutation frequencies of both G:C to A:T transitions at the CpG site of codon 248 and C:G to T:A transitions at codon 247 were observed in colon from UC cases when compared with normal adult controls (P = 0.001 and P = 0.001, respectively). In the UC cases, higher p53 codon 247 and 248 mutation frequencies were observed in the inflamed lesional regions when compared with the nonlesional regions of their colon (P < 0.001 and P = 0.001). The colonic nitric oxide synthase-2 activity was higher in UC cases than in non-UC adult controls (P = 0.02). Our data are consistent with the hypothesis that a higher frequency of p53 mutant cells can be generated under oxidative stress in people with UC. The increased frequency of specific p53 mutated alleles in noncancerous UC colon tissue may confer susceptibility to the development of CRC in an inflammatory microenvironment.

Nitric oxide synthase, cyclooxygenase 2, and vascular endothelial growth factor in the angiogenesis of non-small cell lung carcinoma.

We have investigated the hypothesis that nitric oxide synthase (NOS2), cyclooxygenase-2 (COX2), and vascular endothelial growth factor (VEGF) protein levels individually demonstrate a direct correlation with microvessel density (MVD) and clinical outcome in human non-small cell lung cancer (NSCLC). Furthermore, we hypothesized that MVD may explain the propensity of certain histological lung cancer subtypes for early metastasis via a hematological route. Immunohistochemically, we studied the protein expression levels of NOS2, COX2, and VEGF and MVD by counting CD31-reactive blood vessels (BVs) in 106 surgically resected NSCLC specimens. NOS2, COX2, and VEGF immunoreactivity were observed in 48, 48, and 58%, respectively, of the study subjects, and their levels correlated with MVD at the tumor-stromal interphase (P < or = 0.001). More adenocarcindmas and large cell carcinomas displayed overexpression of NOS2 when compared with squamous cell carcinoma (SCC; r = 0.44; P < 0.001). NOS2 and COX2 levels were found to correlate positively with VEGF status (r = 0.44; P < 0.001, 0.01, and 0.03, respectively). These results attest to the significant interaction of these factors in the angiogenesis of NSCLC. Although neither angiogenic factors nor MVD correlated with patient survival, the latter correlated with tumor clinical stage in both squamous (SCC; 73 BVs/mm2) and non-SCC (78 BVs/mm2) tumors. These results indicate that angiogenesis is a complex process that involves multiple factors including NOS2, COX2, and VEGF. Furthermore, the role of angiogenesis in the biology of various histological lung cancer types may be different. The complexity of angiogenesis may explain the modest results observed in antiangiogenesis therapy that target a single protein.

p53 tumor suppressor gene mutations in transformed cutaneous T-cell lymphoma: a study of 12 cases.

The transformation of cutaneous T-cell lymphoma (t-CTCL) is an uncommon phenomenon that is associated with histopathologic changes and follows an aggressive course. The factors contributing to this transformation are poorly understood. The aim of this study was to analyze the p53 status in t-CTCL and to correlate it with disease outcome. The p53 status was investigated by immunohistochemistry, single-strand conformation polymorphism (SSCP) and DNA sequencing in 12 patients with t-CTCL. Eight mutations were detected; including four in exon 5, one in exon 6 and three in exon 7. Five were point mutations and three were deletions. Paired samples from nontransformed patch and plaque lesions showed no p53 over-expression. Eight disease-related deaths were reported, six to 23 months after transformation, all of which had p53 mutations. Three other patients with wild phenotype (WT-p53) were last reported alive with the disease 19-33 months after transformation (p < 0.0002). One other case had a p53 mutation but a short period of follow-up. Our results suggest that phenotypic changes of t-CTCL are frequently associated with genotype alterations in the p53 gene. Because 70% of the mutations detected were either G to C transversions or deletions, nucleotide-pairing mismatch and not DNA damage by UVB represents a likely mechanism for mutagenesis. Furthermore, the data may help in the design of gene transfer therapies that target the p53 molecule.

Atypical cutaneous lymphocytic infiltrate and a role for quantitative immunohistochemistry and gene rearrangement studies.

AIM: To help clarify the significance of the T-cell receptor (TCR) gene rearrangement and its relationship to the immunophenotyping of histologically atypical cutaneous T-cell lymphoid infiltrates (ACLIs). MATERIALS AND METHODS: One hundred and twenty-four patients presented with lesions clinically suspicious for cutaneous T-cell lymphoma (CTCL). The average age was 55.8 years with a mean follow-up duration of 26.2 months. Cases were classified as malignant (64 cases), inflammatory dermatosis (28 cases), and indeterminate (32 cases), based on follow-up data and histopathology. Quantitative immunophenotyping with computer-assisted imaging was performed using immunohistochemical stains of anti-CD3, CD4, CD5, CD7, CD8, CD20, CD30, CD56, CD68, Bcl-2, p53, and proliferating cell nuclear antigen (PCNA). RESULTS: Abnormal immunophenotypic expression in 87.5% of the malignant cases, including CD4 or CD8 predominance (67%), deletion of pan-T-cell antigens (16.1%), and activation of antigen/oncogene expression (47%), was observed. In addition, 36 clinically malignant cases displayed rearranged bands by polymerase chain reaction (PCR) with TCR beta and gamma. Two benign cases displayed abnormal immunophenotype and two others showed rearranged bands. All of these patients responded to topical steroid therapy with complete resolution. Nineteen indeterminate cases displayed either rearranged bands or immunophenotypic abnormalities, 15 of which were reclassified as malignant. All but three patients improved after CTCL treatment. CONCLUSION: Quantitative immunophenotyping and gene rearrangement analysis can provide detailed information for classifying ACLIs with 91% diagnostic sensitivity and 87% specificity.

p53, c-erbB2, and PCNA status in benign, proliferative and malignant ovarian surface epithelial neoplasms: a study of 75 cases.

Low malignant potential tumors of the ovary are believed to behave in a manner intermediate to their benign and malignant counterparts. However, recent evidence suggests these lesions are in fact benign and better classified as proliferative. Based on our previous work and evaluating p53, c-erbB2, and PCNA status in a full spectrum of ovarian surface epithelial tumors, with emphasis on low malignant potential tumors, we tested this hypothesis. Immunohistochemical stains with monoclonal antibodies were used on 75 archival ovarian neoplasms. The results demonstrated anti-p53 reactivity in 30 carcinomas (40%), 2 of which were proliferative, and no reactivity in the benign tumors. Overexpression of c-erbB2 was seen in 31 malignant neoplasms (64.5%), 4 of which were proliferative (22.1%), and none in benign tumors. The PCNA proliferative index showed means of 42.8%, 22.8%, and 14.9% with benign, low malignant potential, and malignant tumors, respectively. Predicting immunoreactivity in carcinomas for anti-PCNA (Student t test), anti-p53, and anti-c-erbB2 (Pearson chi2 test) versus a lack of immunoreactivity in proliferative tumors indicate P values of .001, <.001, and <.001, respectively. These data show significant differences in the expression of these markers in ovarian tumors and suggest a possible role for these oncogenes as supplemental tools in diagnostic pathology. Further, our findings also support the designation of proliferative as opposed to the current nomenclature of low malignant potential tumors.

Enhancement of tumor killing using a combination of tumor immunization and HSV-tk suicide gene therapy.

Tumor cells genetically modified with the herpes simplex virus thymidine kinase (HSV-tk) gene in combination with ganciclovir (GCV) demonstrate a "bystander effect". Previous attempts to enhance the bystander tumor killing by combining cytokine genes with HSV-tk/GCV have met with varying results. The present study was designed to determine the effects of tumor immunization in combination with HSV-tk gene-modified tumor cells and GCV on tumor killing and to determine if the bystander tumor killing could be enhanced. Tumor-bearing mice immunized with syngeneic tumor (KBALB) prior to treatment with an i.p. injection of xenogeneic HSV-tk gene-modified tumor cells (PA-1STK) had prolonged animal survival (group 4, 56.4 days). In contrast, unimmunized tumor-bearing mice (group 2) or tumor-bearing mice immunized to the xenogeneic PA-1STK tumor cells (group 5) showed a mean survival of about 27 days after receiving an i.p. injection of PA-1STK cells and GCV. Control groups, which were either not immunized and did not receive HSV-tk cells (group 1) or immunized but treated only with GCV (group 3) showed short survival (16-18 days). Analysis of tumors for cytokine mRNA expression revealed increased TNF-alpha and IL-1alpha mRNA expression in group 4 mice. Furthermore, IL-2 mRNA expression was detectable on days 2 and 4 only in group 4 mice. Immunophenotypic analysis for tumor-infiltrating lymphocytes demonstrated an increase in macrophage (4%, p = 0.0001) and T cells (1.8%, p < 0.001) in group 4 mice with an enhanced T-cell response as compared with mice from groups 1, 2 and 3. Our results demonstrate that tumor immunization combined with HSV-tk/GCV treatment results in increased animal survival with enhanced immune response. Furthermore, the cytokine milieu observed in the present study can modulate the tumor micro-environment in vivo from one that is immunosuppressive to one that is immune-stimulatory.

Cancer-prone oxyradical overload disease.

Oxyradical overload disease develops in conditions involving chronic inflammation and may be of inherited etiology, e.g. haemochromatosis and Wilson disease, be acquired, e.g. infection with hepatitis B or C virus or Helicobactor pylori, or be chemically induced, e.g. acid reflux in Barrett oesophagus. Susceptibility to cancer is frequently a pathological consequence of extensive oxyradical damage that leads to a cycle of cell death and regeneration and causes mutations in cancer-related genes. In this brief review, we focus on the possible interactive effects of nitric oxide and the p53 tumour suppressor gene in human carcinogenesis.

Gene therapy for malignant mesothelioma: a novel approach for an incurable cancer with increased incidence in Louisiana.

Malignant mesothelioma (MM) is a tumor of the pleura for which there is no satisfactory treatment. It is an almost universally fatal disease, regardless of the stage of the tumor at the time of diagnosis. Current treatment modalities include surgery, chemotherapy, and radiation therapy, although in some series none of these modalities is superior to no treatment at all. Because of the dismal prognosis for patients with MM, new modes of treatment are desperately needed. A promising area of research into the treatment of various malignancies is gene therapy. Recent studies have demonstrated the utility of exposing tumor cells to cells transduced to express the Herpes simplex virus gene for thymidine kinase (HSV-TK). By virtue of their expression of HSV-TK, the transduced cells are rendered susceptible to the antiviral drug, ganciclovir (GCV). Nearby untransduced tumor cells are killed by a so-called bystander effect. We are describing a Phase I clinical gene therapy trial for MM, which we are presently conducting at the Louisiana State University Medical Center of New Orleans. The purpose is to study the safety and to determine the maximal tolerated dose of an HSV-TK-transduced ovarian cancer cell line (PA1-STK cells) that is infused into the pleural cavities of patients. This infusion is followed by systemic administration of GCV. The hope is that administration of GCV will result in killing of both the transduced ovarian cancer cells as well as the nearby malignant cells.

p53 molecule as a prognostic marker in human malignancies.

The tumor suppressor gene, p53, is the most commonly mutated gene associated with cancer. Mutation of p53 plays a critical role in the multiple stages of carcinogenesis. The functional inactivation of p53 by missense mutations has been described in various cancers and the majority of these mutations occur in exons 5 through 9 of the p53 gene. Mutations leading to the overexpression of p53 have been found to affect the patient survival outcome in several human malignancies. Our experience with more than 200 samples of breast and prostate carcinoma is presented. Our results strongly suggest that the type and location of the p53 mutations within the molecule may affect and dictate the outcome of cancer.

Immunophenotyping as a diagnostic tool to differentiate lichen planus from chronic graft-versus-host disease: diagnostic observations on two patients.

BACKGROUND: Lichen planus (LP) and the lichenoid variant of chronic graft-versus-host disease (cGVHD) can present with similar clinical and histological findings. The distinction, although difficult, is important both prognostically and therapeutically. The mechanism and effector cell phenotypes have also shown to differ between the 2 entities. While the lichenoid infiltrate of LP is predominantly T lymphocytes helper/inducer cell phenotype, the suppressor/cytotoxic subset appears to play a major role in cGVHD. The aim of this study is to determine whether the immunophenotypic character of the lichenoid infiltrate can aid in distinguishing the 2 entities. METHODS: Biopsies were obtained from 2 patients with lichenoid papules and a history of transplantation. Light microscopy revealed lichenoid inflammation in both cases characterized by a band-like lymphohistiocytic infiltrate at the dermal-epidermal junction. Immunochemistry was performed on fresh tissue using a panel of monoclonal antibodies including anti-CD1a, CD3, CD4, CD8, CD16, CD20, CD28, and CD68. Results were quantitated using computer-assisted image analysis. RESULTS: We found that in both cases the majority of cells stained with pan T cell marker CD3+. One case demonstrated predominantly CD4+ T cells and increased numbers of CD1a positive Langerhans cells, while the lymphokine natural killer cell activity (LAK) markers anti-CD16 and anti-CD28 were largely nonreactive. Conversely, the second case contained predominately CD8+ lymphocytes and very few CD1a positive Langerhans cells with abundant LAK cell anti-CD16 and anti-CD28 reactivity. CONCLUSIONS: Based on these findings, the former was classified as lichen planus and the latter as lichenoid cGVHD. The diagnoses are substantiated with clinical history and follow-up information. We conclude that immunophenotypic characteristics of the infiltrate can be a useful tool in differentiating lichenoid cGVHD from lichen planus.

Study of tumor infiltrating lymphocytes and transforming growth factor-beta as prognostic factors in breast carcinoma.

Cytokines and growth factors are powerful modulators of the immune response. Their aberrant expression either by the tumor cells or by the tumor infiltrating lymphocytes confers a selective advantage to the tumor to grow and suppress the cytotoxic activity of the infiltrating lymphocytes. Therefore, analysis of these soluble factors in the tumor microenvironment can provide an insight into the understanding of the tumor behavior and may be used as a prognostic factor. In the present study the nature of the tumor infiltrating lymphocytes (TILs) and cytokine profile was examined in 36 and 19 mammary carcinoma tissues, respectively, by immunohistochemistry and PCR. Phenotypic differences in the number of cytotoxic T lymphocytes (CD8+) and lymphokine activated killer cells (CD16) was observed among TILs when patients with either early disease stage (39% and 46.6%, respectively) or those alive with no residual disease (31% and 52%, respectively) were compared with late stage (9.7% and 22.8%, respectively) or those dead of disease (14.6% and 15.6%, respectively). Furthermore, analysis of the 19 tumor samples for cytokine mRNA expression by RT-PCR revealed the presence of TNF-alpha, IL-10, TGF-beta1, and IL-2. However, semi-quantitative PCR analysis demonstrated TGF-beta1 expression to be significantly higher in patients with a favorable outcome (1.0246 attomoles/micromoles) as compared to patients with a poor prognosis (0.1157 attomoles/micromoles). Our results demonstrate the potential biological significance of certain host factors, particularly TILs and TGFbeta1 expression, on the outcome of breast cancer.

DNA ploidy and proliferating cell nuclear antigen image analysis of peritoneal and pleural effusions. A possible diagnostic role.

OBJECTIVE: To determine the role of DNA and proliferating cell nuclear antigen (PCNA) image analysis (IA) in enhancing the diagnostic sensitivity of conventional cytology (CC). STUDY DESIGN: The histopathologic and clinical data on 87 consecutive pleural and peritoneal effusions were used to evaluate the accuracy of CC and DNA IA results. RESULTS: CC showed a sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 65%, 100%, 100% and 62%, respectively. Aneuploidy peaks were seen in 49 cases; 47 of them were true positives. Thirty of 38 diploid cases were true negatives. The sensitivity, specificity, PPV and NPV were 85%, 94%, 96% and 80%, respectively. There were positive correlations between DNA ploidy profile and PCNA proliferative index (PI), (R = .697) and significant differences in PCNA PI between malignant and benign effusions (P < .001). CONCLUSION: The DNA IA PI by PCNA can be used as a complementary diagnostic tool with CC in cytologically inconclusive cases.

Tumor-host interaction: analysis of cytokines, growth factors, and tumor-infiltrating lymphocytes in ovarian carcinomas.

The host-tumor interaction may play an important role in determining tumor progress. Recent studies have shown that this interaction can be influenced by the release of soluble factors by tumor cells and tumor-infiltrating lymphocytes (TIL). The aim of our study is to characterize the nature of cytokines and growth factors and their relationship to the cellular infiltrates in 16 patients with ovarian cancer using reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. Total RNA from 20 malignant and 10 benign specimens were used to assay for expression of 12 cytokines. Additionally, monoclonal antibodies (MAbs) were used to detect T cells, CD4+ helper and CD8+ cytotoxic/suppressor T-cell subtypes, B cells, and macrophages. Our results showed the expression of transforming growth factor-beta1 (TGF-beta1), interleukin-10 (IL-10), and granulocyte-macrophage colony-stimulating factor (GM-CSF) in 19, 17, and 10 malignant specimens, P < .001, .001, and .05, respectively. Other cytokines such as interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), TNF-beta/LT, IL-2, and IL-6 were expressed in a few cases, and IL-1alpha and IL-4 expression were not detected. The benign samples did not express IL-10, but GM-CSF, TGF-beta1, and IL-8 were expressed in one, one, and four specimens, respectively. Interestingly, in four cases in which samples from the primary and relapse tumors were available for analysis, the tumors in relapse showed a significant increase for TGF-beta1 (P < .05) and a decreased trend in IL-10 mRNA levels. The source of these factors was tumor cells as detected immunohistochemically. This combined alteration of TGF-beta1 and IL-10 was associated with a significant reduction in number of TIL in general, and CD8+ and macrophages in particular (P = .036 and .049, respectively). Our findings suggest the important role of certain soluble factors in the complex process of tumor progression. Furthermore, understanding the tumor-host relationship and the factors influencing the interaction may be helpful in developing effective and innovative treatment methods.

An immunohistochemical study of leu 7 and PCNA expression in thyroid neoplasms.

Monoclonal Antibody (MoAb) HNK, or anti-leu-7, is reactive with several neuroendocrine and nonneuroendocrine tumors. The aim of this study is to examine anti-leu-7 reactivity in thyroid neoplasms and its relationship to cellular proliferation as determined by anti-PCNA reactivity. The expression of anti-leu-7 in 56 thyroid neoplasms (24 papillary carcinomas, 14 follicular carcinomas, two medullary carcinomas and 16 follicular adenomas) was examined immunohistochemically. Papillary and follicular thyroid carcinomas reacted with anti-leu-7 in a membranous and cytoplasmic pattern in 88% and 93% of cases, respectively. The adjacent benign tissues were nonreactive. Only eight cases diagnosed as follicular adenomas were reactive with anti-leu-7. Furthermore, the mean proliferative index (PI), as measured by the percentage of nuclei immunoreactive with anti-PCNA, was greater than 30% in all thyroid neoplasms reactive with anti-leu-7. The PI was 58% for papillary carcinomas and 68% and 48% for follicular carcinomas, and follicular adenomas, respectively. Lesions originally classified as follicular adenomas that were nonreactive with anti-leu-7 had a PI of 24% and were reclassified as hyperplastic nodules. These data suggest that anti-leu-7 may be useful for characterizing thyroid neoplasia.