Oxytocin receptor pattern of expression in primary lung cancer and in normal human lung

In order to assess if oxytocin- and vasopressin-induced mitogenic effects detected on small-cell lung carcinoma (SCLC) cell lines could be transposed on primary SCLC, the aim of the present work was to identify mediators of these mitogenic actions on primary tumours samples. This was addressed on normal human lung tissue, on SCLC and on non-SCLC (NSCLC). Herein, we observe, in normal human lung, that OTR is colocalized with vascular endothelial cells of the lung and is not expressed by lung cells of epithelial nature. We detected mRNA amplification of V1aR, V2R and of a V2R variant. We observed that 86% of SCLC biopsies analyzed expressed at least the OTR and that 71% expressed the OTR, the V1aR and the V2R altogether. Comparatively, 50% of NSCLC biopsies tested expressed at least the OTR and 32% expressed the OTR, the V1aR and the V2R altogether. The occurrence of the V1bR/V3R is of 28 and 18% for SCLC and NSCLC, respectively. Nevertheless, for the SCLC biopsies analyzed in this study, V1bR/V3R expression correlates, in all cases, with the expression of all the other neurohypophysial peptide receptors. Our results suggest that neurohypophysial peptide antagonists may offer promise as a potential new therapeutic modality for the treatment of lung cancer expressing at least one of the neurhypophysial peptide receptor subtypes.     

Vitamin D receptor expression in normal, premalignant, and malignant human lung tissue.

BACKGROUND: There is a strong interest in identifying chemopreventive agents that might help decrease the burden of lung cancer. The active metabolite of vitamin D, 1,25-dihydroxycholecalciferol (calcitriol), has been shown to have antiproliferative effects in several tumor types, mediated by the vitamin D receptor (VDR). This is the first comprehensive survey of VDR expression in a series of human lung tissues, including normal and premalignant central airway biopsies and lung tumors. METHODS: Immunohistochemical expression of nuclear and cytoplasmic VDR was examined in 180 premalignant or malignant bronchial biopsies from bronchoscopy of 78 high-risk individuals at the Roswell Park Cancer Institute and also in 63 tumor samples from 35 lung cancer patients from the University of Chicago Hospitals. Associations between clinicopathologic data and VDR expression were examined. RESULTS: VDR expression was present in many samples. In biopsies, VDR was commonly detected throughout the full epithelial layer. Most histologically normal (60%, 53 of 88) and metaplastic (61%, 39 of 64) samples had moderate to high nuclear intensity; dysplastic samples mostly had low nuclear intensity (10 of 18, 55%). In tumor samples, 62% (38 of 61) were lacking cytoplasmic VDR, with nuclear expression present in 79%(49 of 62). Analysis of all samples revealed a positive linear trend between proportion of samples with greater nuclear than cytoplasmic intensity and increasing histologic grade (P < 0.01). CONCLUSIONS: VDR expression spanned the lung carcinogenesis spectrum. Nuclear expression was similar across various histologies, whereas cytoplasmic expression decreased with increasing histologic grade. These results indicate that there is potential for the use of calcitriol as a chemopreventive agent against the development of lung cancer.     

Interleukin 4 receptor expression and growth inhibition of gastric carcinoma cells by interleukin 4.

The expression of the interleukin 4 (IL-4) receptor (IL-4R) and effects of human recombinant IL-4 on human gastric carcinoma cell lines were studied. We demonstrated that IL-4 inhibited the growth of gastric carcinoma cells in a dose dependent manner (0.1-100 units/ml) in a [3H]thymidine incorporation proliferation assay. The gastric carcinoma cells varied in sensitivity to treatment with low dose IL-4. Treatment of cells with IL-4 altered the morphology of the cells to a "flattened" morphological shape resembling differentiation. The IL-4-mediated growth inhibition was significantly abrogated by neutralization of IL-4 with specific anti-IL-4 antibody. IL-4R expression on the cell surface was determined by assessing biotin-labeled IL-4 binding to cells using flow cytometry. IL-4R expression ranged from 5 to 85% of total cell population in the gastric carcinoma cell lines assessed. There was a positive correlation between the sensitivity to IL-4-mediated growth inhibition and IL-4R expression. By Northern blot analysis, we demonstrated that mRNA of IL-4R was expressed in the gastric carcinoma cells. Using in situ hybridization, we confirmed that IL-4R mRNA was expressed in the gastric carcinoma cell at the single cell level. By using a sensitive polymerase chain reaction technique, we demonstrated that gastric carcinoma cells expressed IL-4 mRNA, suggesting a possible autocrine loop. These studies indicate that IL-4 can significantly modulate gastric carcinoma cells that possess IL-4R. IL-4R on gastric carcinoma cells may be a potential therapeutic target site for IL-4-directed therapy.     

VEGF receptor expression and signaling in human bladder tumors

Overexpression of vascular endothelial growth factor receptors (VEGFRs) has been reported in a variety of tumor types. Here we find that 11 out of the 14 bladder tumor cell lines examined express one or more VEGF receptors. Analysis of the T24 bladder tumor cell line reveals a functional autocrine loop involving VEGF and the Flk-1 receptor. Blocking VEGF expression in T24 cells results in a decrease in DNA synthesis. The Flk-1 receptor in T24 cells is phosphorylated in response to VEGF-121 or VEGF-165, and an Flk-1 inhibitor blocks VEGF to ERK signaling. We report that VEGF stimulation of T24 cells results in activation of H- and N-Ras and this is dependent on cellular sphingosine kinase 1 (SPK1) activity. Previously, we found VEGF-induced activation of Ras appears to be independent of a Ras-guanine nucleotide exchange factors (GEFs). Here we report that sphingosine can stimulate Ras-GTPase activating protein (GAP) activity in vitro, and sphingosine-1-phosphate (SPP) can block the stimulatory effects of sphingosine. We present a model where the balance between sphingosine and SPP regulates Ras-GAP activity such that stimulation of SPK1 favors downregulation of Ras-GAP and thereby the activation of Ras proteins. These data highlight a VEGF pathway that may be involved in the survival and proliferation of bladder tumor cells as well as other tumor cell types.     

Immunohistochemical expression of cyclooxygenase isoenzymes and downstream enzymes in human lung tumors.

PURPOSE:Prostanoids are important mediators of pulmonary vaso- and bronchotone regulation and strongly influence inflammatory reactivity. The product of cyclooxygenase (Cox), prostaglandin H(2), is further metabolized via downstream enzymes into the different effective metabolites. The specific cellular equipment with certain downstream enzymes crucially determines the cellular reactivity by generation of functionally different prostanoid metabolites. EXPERIMENTAL DESIGN: To elucidate the role of arachidonic acid metabolism via the cyclooxygenase pathway in different human lung tumors, expression of cyclooxygenase isoenzymes (Cox-1 and Cox-2) and downstream enzymes of prostanoid metabolism was investigated in human non-small cell lung cancer and normal human lung tissue by immunohistochemical techniques. RESULTS: In comparison to strong Cox-1 reactivity in airways and endothelial cells of normal lung specimens, only 4 of 15 adenocarcinomas showed infrequent Cox-1 expression. All lung cancer specimens displayed an increased Cox-2 immunostaining pattern with strong reactivity in adenocarcinomas and lower reactivity in squamous cell carcinomas. Adenocarcinomas and squamous cell carcinomas were also positive for thromboxane A(2) synthase, prostaglandin D(2) synthase, and prostaglandin E(2) synthase, but not for prostacyclin synthase. Endothelial cells of vessels found within or near the tumor show extensive immunostaining of Cox-2 and thromboxane A(2) synthase, whereas endothelial cells of normal lung specimens, in contrast, expressed Cox-1 and prostacyclin synthase. CONCLUSIONS: We conclude that non-small cell lung cancer shows a specific Cox-/downstream-enzyme expression pattern, which is specifically altered in lung tumor cells and tumor supplying vessels in contrast to normal lung tissue. This may have major impact on tumor progression and tumor-associated inflammation via an altered prostanoid metabolism with consecutive tumor-associated blood flow distribution.     

Evaluation of NQO1 gene expression and variant allele in human NSCLC tumors and matched normal lung tissue

NQO1 gene expression was evaluated by RT-PCR and SNP status by RFLP in matched samples of lung tumors and adjacent normal tissue. NQO1 was found to be overexpressed in lung tumors when compared to matched normal lung tissue. The mean expression in normal lung tissue was 28.26 x 10(-14) ng/microl +/- 44.9 SD and 61.46 x 10(-14) ng/microl +/- 103.2 SD in lung tumors. NQO1 gene expression was higher in the tumor than in the matched normal lung tissue in 27/50 (59%) patients (p=0.014). In the normal samples, 25 (50%) were wild-type, 16 were heterozygotes (32%) and 8 had the SNP (16%). In the matched tumor samples 14 were wild-type (28%), 16 were heterozygous (32%) and 19 (38%) had the SNP (p=0.0043). The genomic NQO1 mutation was associated with survival in a pilot study of stage II/III NSCLC patients. Patients with a homozygous SNP genotype had a significantly shorter survival (median 12 months), than heterozygous or homozygous wild-type patients (median 41 months) (p=0.007), suggesting NQO1 may be important in chemosensitivity as well as the pathogenesis of lung cancer and NQO1 genotyping may be a useful component of pharmacogenetic strategies for the treatment of NSCLC.     

Human lung organ-specific antigens on normal lung, lung tumors, and a lung tumor cell line.

An antiserum raised in rabbits against a lung tumor cell line (2563) was selected from a library of antisera against normal and malignant human lung, lung tumor cell lines, and fetal tissues and was found by complement fixation, immunofluorescence, and saturation binding assays to contain antibodies for antigens characteristic of those found in normal lung. Studies with the adsorbed antiserum (A49) revealed: 1) An antigen was shared by normal lung and normal kidney (NLK-1) 2) lung tissue-specific antigen(s) were present on normal lung tissue (NL-1); 3) NL-1 was found on both external and internal cell membranes; and 4) NL-1, in addition to being present on normal lung and the adenocarcinoma-derived cell line 2563, was present on 1 of 2 metastatic lung adenocarcinomas but on none of 4 metastatic lung tumors of other histologic types.     

Cloning of 67-kDa laminin receptor cDNA and gene expression in normal and malignant cell lines of the human lung.

Cell-adhesive protein laminin and its specific receptor play an important role in the processes of cancer proliferation, invasion and metastasis. In the present study, we cloned the cDNAs of the 67-kDa laminin receptor both from a human lung cell line (IMR90) and from a human lung cancer cell line (SBC3), and determined the nucleotide sequences. In comparison with both cDNA sequences of the protein-coding region, three nucleotide differences were found. These differences in the secondary structure of the protein, however, were caused by nucleotide substitutions. It was also demonstrated that the level of 67-kDa-laminin receptor mRNA was higher in SBC3 than in IMR90.     

Cadherin and catenin expression in normal human bronchial epithelium and non-small cell lung cancer.

Cadherins are transmembrane cell adhesion molecules (CAMS) that mediate cell-cell interactions and are important for maintenance of epithelial cell integrity. This function is dependent on an indirect interaction between the cytoplasmic domain of the cadherin molecule with three cytoplasmic proteins known as alpha-, beta-, and gamma-catenin (-cat). Growing evidence suggests that alterations in cadherin or catenin expression or function may be important to the development of an invasive or metastatic phenotype. Immunohistochemical techniques were used to study the expression of the two major epithelial cadherins, E-cadherin (E-cad) and P-cadherin (P-cad) as well as alpha- and gamma-cat in normal bronchial epithelium and in a series of carefully TMN-staged pulmonary adenocarcinomas (n = 21) and squamous cell carcinomas (n = 7). The cadherin profile of normal pseudostratified bronchial epithelium was heterogeneous. Basilar cells strongly expressed P-cad, alpha- and gamma-cat, while columnar cells moderately expressed E-cad, alpha- and gamma-cat. In contrast to other epithelial tumors, E-cad on non-small cell lung carcinomas was actually upregulated, however, a decrease in P-cad expression was noted in 68%. At least one cadherin or catenin was downregulated, compared to normal bronchial epithelium, in 82% of tumors examined. With the exception of an association between loss of P-cad expression and poorly differentiated state, changes in cadherin and catenin expression levels were not significantly correlated to tumor stage, cell type, or nodal status. These findings illustrate that alteration of expression of cadherins and catenins are often found in non-small cell lung carcinoma when compared to the progenitor bronchial epithelium, and may play a role in the development of the malignant phenotype.     

趋化因子受体4与肿瘤

趋化因子受体4（CXCR4）属于G蛋白耦联受体超家族,具有趋化免疫细胞、维持免疫细胞的动态平衡等生物学作用。CXCR4表达于多种类型的组织和细胞,在多种肿瘤及肿瘤的不同阶段,CXCR4的表达都明显高于正常组织。CXCR4与肿瘤细胞的增殖、黏附、侵袭和转移有关,在肿瘤的进展中发挥重要作用。     

趋化因子受体4与肿瘤

趋化因子受体4（CXCR4）属于G蛋白耦联受体超家族,具有趋化免疫细胞、维持免疫细胞的动态平衡等生物学作用。CXCR4表达于多种类型的组织和细胞,在多种肿瘤及肿瘤的不同阶段,CXCR4的表达都明显高于正常组织。CXCR4与肿瘤细胞的增殖、黏附、侵袭和转移有关,在肿瘤的进展中发挥重要作用。     

Flow cytometric analysis of androgen receptor expression in human prostate tumors and benign tissues.

Androgen receptor (AR) plays an important role in growth and hormonal therapy of human prostate tumors. Immunohistochemical analysis of AR expression, a nonquantitative technique, is currently used for screening of receptor expression in prostate tissues. The present report describes a laser flow cytometric method for monitoring AR expression in human cell lines and in archival formalin-fixed paraffin-embedded prostate tissues and tumors. Multiparametric flow analysis can be used for simultaneous detection of other cellular markers (e.g., DNA aneuploidy), and by gated analysis, AR expression in subpopulations of a tumor can be quantitatively determined.     

p53 mutations in human lung tumors.

Mutation of one p53 allele and loss of the normal p53 allele [loss of heterozygosity (LOH)] occur in many tumors including lung cancers. These alterations apparently contribute to development of cancer by interfering with the tumor suppressor activity of p53. We directly sequenced amplified DNA in the mutational hot spots (exons 4-8) of p53 in DNA samples from 40 lung cancers. Most (31 of 40) samples were preselected for LOH in the region of p53. We detected 23 p53 mutations within these exons in 22 lung cancers; no p53 mutations were found in normal tissue of the patients. One-half of the mutations were G to T transversions on the nontranscribed strand, consistent with mutagenesis by tobacco smoke. Mutations of C to A on the nontranscribed strand, which would result from G to T mutations on the transcribed strand, were detected only in one sample. Three of 23 mutations were nonsense mutations; to date, nonsense mutations of p53 have not been reported in lung cancer. Mutation of this p53-coding region was detected in 20 of 27 small cell lung cancer samples, representing a 70% occurrence. Mutation of the p53 gene is apparently very frequent in small cell lung cancers. When LOH in the p53 region could be determined, complete concordance occurred between a sample having both a p53 mutation and LOH in the region of p53 (18 of 18 samples). Twelve samples of lung cancer had LOH in the region of p53, but the samples had no detectable p53 mutations, suggesting either alterations outside the known mutational hot spots of p53 or alterations of another unidentified tumor suppressor gene in the region of p53.     

The expression of placental glutathione-s-transferase (gst-pi) in human normal salivary-glands and tumors

Immunohistochemical localization of placental glutathione S-transferase (GST-pi) in normal and inflammatory salivary glands (74 cases) and tumors (71 cases) was studied. In the normal and inflamed salivary glands of all locations (including major and minor salivary glands), the ductal epithelial cells showed moderate to strong GST-pi staining, myoepithelial cells showed weak staining and the acinar cells were negative. The staining pattern of the tumor cells was similar to that of their normal counterparts. The tumor cells were generally positive with GST-pi staining except those tumor cells demonstrating acini differentiation (serous cells in acinic cell carcinoma, mucous cells in mucinous cystadenoma and mucoepidermoid carcinomas). Most of the salivary gland tumors, benign or malignant, showed a weak GST-pi staining. Only mucoepidermoid carcinomas demonstrated significantly increased GST-pi reactivity compared to other tumors (p<0.0001), which may be a reflection of both malignancy and squamous differentiation of the tumor. The marked increase in GST-pi activity in mucoepidermoid carcinomas may be useful in serological screening of recurrent and metastatic mucoepidermoid carcinomas.     

Correlation between c-erbB-4 receptor expression and response to gemcitabine-cisplatin chemotherapy in non-small-cell lung cancer.

Background:While the overexpression of c-erbB gene family inseveral malignancies is associated with poorer prognosis, the associationbetween the expression of the cellular markers and the response tochemotherapy is not yet clear. In this study we investigated the expressionof c-erbB-4 receptor in NSCLC and correlated it with the response togemcitabine–cisplatin combination chemotherapy. Patients and methods:Forty-three NSCLC patients withhistologically or cytologically proven disease were treated withgemcitabine–cisplatin combination chemotherapy. Immunohistochemicalstains for c-erbB-4 receptor were performed in 20 cases on paraffin sectionsusing the avidin-biotin-peroxidase method. Results:Two patients achieved complete response (5%), and16 achieved partial response (37%) yielding an overall objectiveresponse rate of 42%. Minimal response was observed in seven patients(16%) and disease stabilization in 7%. Immunohistochemical stainwas positive for the presence of c-erbB-4 receptor in 25% of patients,and negative in 75%. No response was documented in c-erbB-4 positivepatients (0 of 5) while an objective response (complete, partial orminimal) was seen in 11 of 15 (73%) c-erbB-4 negative patients.Negative stain for c-erbB-4 significantly favored response togemcitabine–cisplatin combination (P < 0.01). Conclusion:C-erbB-4 expression status showed no correlation withsurvival and cannot be accepted at this time as a guiding factor fortherapeutic management. These interesting results deserve further evaluationin a large-scale prospective trial before treatment recommendations on thebasis of c-erbB-4 presence can be finally made.     

Prevalence of KIT expression in human tumors.

PURPOSE: KIT is a target for imatinib mesylate (Gleevec; Novartis Pharma, Basel, Switzerland). Gastrointestinal stromal tumors (GISTs) express KIT and respond favorably to imatinib therapy. To determine other tumors in which such a molecular targeted therapy might be indicated, we investigated KIT expression in different human tumor types. Because recent studies in GISTs suggest that KIT-activating mutations predict response to imatinib therapy, we also sequenced a subset of positive tumors. MATERIALS AND METHODS: More than 3,000 tumors from more than 120 different tumor categories were analyzed by immunohistochemistry in a tissue microarray format. Seven commercially available anti-KIT antibodies were initially evaluated. The antibody A4502 (DAKO) was selected for analysis because of a high frequency of positivity in GIST and low staining background in other tissues. To determine the frequency of KIT mutations in various tumor types, the exons 2, 8, 9, 11, 13, and 17 (where mutations previously were reported) were sequenced in 36 tumors with strong KIT expression. RESULTS: KIT positivity was detected in 28 of 28 GISTs (100%), 42 of 50 seminomas (84%), 34 of 52 adenoid-cystic carcinomas (65%), 14 of 39 malignant melanomas (35%), and eight of 47 large-cell carcinomas of the lung (17%), as well as in 47 additional tumor types. KIT mutations were found in six of 12 analyzed GISTs, but only in one of 24 other tumors. CONCLUSION: The results suggest that KIT expression occurs infrequently in most tumor types and that, with the exception of GISTs, KIT gene mutations are rare in immunohistochemically KIT-positive tumors.