Members of the heat-shock protein 70 family promote cancer cell growth by distinct mechanisms

Whereas the stress-inducible heat-shock protein 70 (Hsp70) has gained plenty of attention as a putative target for tumor therapy, little is known about the role of other Hsp70 proteins in cancer. Here we present the first thorough analysis of the expression and function of the cytosolic Hsp70 proteins in human cancer cells and identify Hsp70-2, a protein essential for spermatogenesis, as an important regulator of cancer cell growth. Targeted knock-down of the individual family members by RNA interference revealed that both Hsp70 and Hsp70-2 were required for cancer cell growth, whereas the survival of tumorigenic as well as nontumorigenic cells depended on Hsc70. Cancer cells depleted for Hsp70 and Hsp70-2 displayed strikingly different morphologies (detached and round vs. flat senescent-like), cell cycle distributions (G2/M vs. G1 arrest) and gene expression profiles. Only Hsp70-2 depletion induced the expression of macrophage inhibitory cytokine-1 that was identified as a target of P53 tumor-suppressor protein and a mediator of the G1 arrest and the senescent phenotype. Importantly, concomitant depletion of Hsp70 and Hsp70-2 had a synergistic antiproliferative effect on cancer cells. Thus, highly homologous Hsp70 proteins bring about nonoverlapping functions essential for cell growth and survival.     

Molecular determinants of human uveal melanoma invasion and metastasis

The molecular analysis of cancer has benefited tremendously from the sequencing of the human genome integrated with the science of bioinformatics. Microarray analysis technology has the potential to classify tumors based on the differential expression of genes. In the current study, a collaborative, multidisciplinary approach was utilized to study the molecular determinants of human uveal melanoma invasion and metastasis. Uveal melanoma is considered the most common primary intraocular cancer in adults, resulting in the death of approximately 50% of patients affected. Unfortunately, at the time of diagnosis, many patients already harbor microscopic metastases, thus underscoring a critical need to identify prognostic markers indicative of metastatic potential. The investigative strategy consisted of isolating highly invasive vs. poorly invasive uveal melanoma cells from a heterogeneous tumor derived from cells that had metastasized from the eye to the liver. The heterogeneous tissue explant MUM-2 led to the derivation of two clonal cell lines: MUM-2B and MUM-2C. Further morphological and functional analyses revealed that the MUM-2B cells were epithelioid, interconverted (expressing mesenchymal and epithelial phenotypes) highly invasive, and demonstrated vasculogenic mimicry. The MUM-2C cells were spindle-like, expressed only a vimentin mesenchymal phenotype, poorly invasive, and were incapable of vasculogenic mimicry. The molecular analysis of the MUM-2B vs. the MUM-2C clones resulted in the differential expression of 210 known genes. Overall, the molecular signature of the MUM-2B cells resembled that of multiple phenotypes – similar to a pluripotent, embryonic-like genotype. Validation of select genes that were upregulated and down-regulated was conducted by semiquantitative RT-PCR measurement. This study provides a molecular profile that will hopefully lead to the development of new molecular targets for therapeutic intervention and possible diagnostic markers to predict the clinical outcome of patients with uveal melanoma. This revised version was published online in July 2006 with corrections to the Cover Date.     

Gene profiling reveals increased expression of uteroglobin and other anti-inflammatory genes in glucocorticoid-treated nasal polyps

BACKGROUND: Treatment with local glucocorticoids (GCs) decreases symptoms and the size of nasal polyps. This might depend on the downregulation of proinflammatory genes, as well as the upregulation of anti-inflammatory genes. OBJECTIVE: We sought to identify GC-regulated anti-inflammatory genes in nasal polyps. METHODS: Affymetrix DNA microarrays were used to analyze the expression of 22,283 genes in 4 nasal polyps before and after local treatment with fluticasone (400 microg/d). Expression of uteroglobin and mammaglobin B was analyzed with real-time PCR in 6 nasal polyps and in nasal biopsy specimens from 6 healthy control subjects. RESULTS: Two hundred three genes had changed in expression in treated polyps, and 139 had known functions: 54 genes were downregulated, and 85 were upregulated. Genes associated with inflammation constituted the largest single functional group. These genes affected key steps in inflammation (eg, immunoglobulin production; antigen processing and presentation; and the chemoattraction and activation of granulocytes, T cells, and B cells). Several proinflammatory genes were downregulated. In contrast, some anti-inflammatory genes were upregulated. The gene that increased most in terms of expression was uteroglobin. This was confirmed with real-time PCR. By contrast, expression of uteroglobin was lower in untreated polyps than in healthy nasal mucosa. Immunohistochemical investigation showed staining of uteroglobin in the epithelium and in seromucous glands in control subjects and in nasal polyps. CONCLUSION: Upregulation of anti-inflammatory genes, such as uteroglobin, might contribute to the effects of local treatment with GCs in nasal polyps.     

Tissue remodeling of rat pulmonary artery in hypoxic breathing. I. Changes of morphology, zero-stress state, and gene expression

The remodeling of the pulmonary arterial tissue in response to a step change of the oxygen concentration in the gas in which a rat lives was recorded as function of time and function of O₂ concentration. Three steps of changing from 20.9% to 17.2%, 13.6%, and 10% O₂ were imposed. Earlier work in our laboratory has shown that pulmonary arterial tissue remodeling is significant in the first 24 h after a step change of oxygen tension. Hence we made measurements in this period. Furthermore, data were obtained for tissue remodeling of circumferential and axial lengths of the pulmonary arteries. We recorded the activities of gene expressions in the lung tissues by microarray, determined the dose response curves of gene expression in the homogenized whole lungs with respect to four levels of O₂ concentration, and obtained the time courses of gene expression in the lung parenchyma in 30 days after a step decrease of O₂ concentration from 20.9% to 10%. We would like to suggest that the correlation of gene expression with physiological function parameters, i.e., time, O₂ tension, blood pressure, opening angle, wall thicknesses, etc., is the way to narrow down the search for specific genes for specific physiological functions. © 2001 Biomedical Engineering Society. PAC01: 8719Uv     

A neural network-based similarity index for clustering DNA microarray data

A common approach to the analysis of gene expression data is to define clusters of genes that have similar expression. A critical step in cluster analysis is the determination of similarity between the expression levels of two genes. We introduce a neural network-based similarity index as a non-linear similarity index and compare the results with other proximity measures for Saccharomyces cerevisiae gene expression data. We show that the clusters obtained using Euclidean distance, correlation coefficients, and mutual information were not significantly different. The clusters formed with the neural network-based index were more in agreement with those defined by functional categories and common regulatory motifs.     

Calretinin expression in human normal and neoplastic tissues: a tissue microarray analysis on 5233 tissue samples

Calretinin is a calcium-binding protein expressed in different normal and neoplastic tissues. Early studies suggested that calretinin is a useful marker to differentiate adenocarcinomas from malignant mesotheliomas of the lung, but subsequent work has shown that calretinin can be expressed in several other tumor types. To systematically investigate the epidemiology of calretinin expression in normal and neoplastic tissues, we used tissue microarrays (TMAs) to analyze the immunohistochemically detectable expression of calretinin in 5233 tissue samples from 128 different tumor categories and 76 different normal tissue types. At least 1 case with weak expression could be found in 74 of 128 (58%) different tumor types and 46 entities (36%) had at least 1 tumor with strong positivity. In normal tissues, a particularly strong expression was found in Leydig cells of the testis, neurons of the brain, theca-lutein and theca interna cells of the ovary, and mesothelium. In tumors, strong calretinin expression was most frequently found in malignant mesotheliomas (6 of 7), Leydig cell tumors of the testis (5 of 5), adenomas of adrenal gland (5 of 9), and adenomatoid tumors (4 of 9). In summary, calretinin is frequently expressed in many different tumor types. Metastases of various different origins must be included in the differential diagnosis of calretinin-positive pleura tumors.     

Transcriptome analysis in blastocyst hatching by cDNA microarray

BACKGROUND: Hatching is an important process for early embryo development, differentiation and implantation. However, little is known about its regulatory mechanisms. By integrating the technologies of RNA amplification and cDNA microarrays, it has become possible to study the gene expression profile at this critical stage. METHODS: Pre-hatched and hatched ICR mouse embryos (25 blastocysts in each group were used in the triplicate experiments) were collected for RNA extraction, amplification, and microarray analysis (the mouse cDNA microarray, 6144 genes, including expressed sequence tags). RESULTS: According to cDNA microarray data, we have identified 85 genes that were expressed at a higher level in hatched blastocyst than in pre-hatched blastocysts. In this study, 47 hatching-related candidate genes were verified via re-sequencing. Some of these genes have been selected and confirmed by real-time quantitative RT-PCR. These hatching-specific genes were also expressed at a lower level in the delayed growth embryos (morula or blastocyst without hatching at day 6 post hCG). These genes included: cell adhesion and migration molecules [E-cadherin, neuronal cell adhesion molecule (NCAM), lectin, galactose binding, soluble 7 (Lgals7), vanin 3 and biglycan], epigenetic regulators (Dnmt1, and SIN3 yeast homolog A), stress response regulators (heme oxygenase 1) and immunoresponse regulators [interleukin (IL)-2-inducible T-cell kinase, IL-4R, interferon-gamma receptor 2, and neurotrophin]. The immunostaining of E-cadherin and NCAM showed strong and specific localization in hatched blastocyst. CONCLUSIONS: This work provides important information for studying the mechanisms of blastocyst hatching and implantation. These hatching-specific genes may have potential as new drug targets for controlling fertility.     

Correlation of AIB1 overexpression with advanced clinical stage of human colorectal carcinoma

AIB1, a member of the steroid receptor coactivator 1 family, has been cloned on 20q12 and is a candidate oncogene in human breast cancer. It is commonly amplified and overexpressed in several types of human cancers. In this study, we examined the expression of AIB1, as related to clinicopathologic features, in 85 human colorectal cancers (CRCs). The status of the number of AIB1 copies, p53 expression, and DNA ploidy was also analyzed. The overexpression of AIB1 was detected in 35% of CRCs. Amplification of AIB1 was observed in 10% of CRCs. In addition, the overexpression of AIB1 was observed more frequently in CRCs in later clinical stages (T3 N1 M0/T3 N0 2M1), compared with that in T3 N0 M0 stage (P < .05). These results suggest that overexpression of AIB1 might provide a selective advantage for the developmental growth and/or progression of subsets of CRCs. In addition, a significant correlation (P < .05) of overexpression of AIB1 with p53 overexpression as well as with aneuploid DNA content was observed in these CRCs. The overexpression of p53 was also correlated significantly with CRC DNA ploidy (P < .05). Furthermore, there was a substantial population of CRCs showing overexpression of both AIB1 and p53 protein and all had aneuploid DNA content; most of these were in the later clinical stage. These findings suggest a possible convergence of AIB1 with a pathway involving p53, which might induce chromosomal instability and affect the clinical phenotype of a subset of CRCs.