Proteomic analysis of human thyroid cell lines reveals reduced nuclear localization of Mn-SOD in poorly differentiated thyroid cancer cells

Differential protein arrays between nuclear extracts of human thyroid cell lines obtained from tumors with different degree of differentiation were exploited to define molecular alterations occurring during thyroid tumor progression. Nuclear extracts from the well differentiated TPC-1 (from papillary carcinoma) and the poorly differentiated ARO (from anaplastic carcinoma) cells showed an overall similar pattern of protein expression as revealed by two-dimensional gel electrophoresis analysis. However, manganese-superoxide dismutase (Mn-SOD) was clearly identified by mass spectrometry procedures as significantly less expressed in ARO compared to TPC-1 cells. A reduced expression of Mn-SOD in the nuclear compartment was confirmed by Western blot and immunofluorescence analysis. A similar expression pattern of nuclear Mn-SOD was detected by immunohistochemistry in human thyroid tumors, with the lowest or absent detection in anaplastic carcinomas. Moreover, the levels of nuclear Mn-SOD in tumor cells were lower than in the normal thyrocytes. These data indicate that an altered nuclear expression of Mn-SOD parallels, together with changes in other elements of the antioxidant protective system, the loss of differentiation occurring during the progression of thyroid tumors.     

Malignant rhabdoid tumor of the colon

PURPOSE: Malignant rhabdoid tumors were first described in the kidney as a rare variant of Wilms' tumor with a “rhabdomyosarcomatoid” pattern and a particularly poor prognosis. Further studies have demonstrated these neoplasms as a distinct clinicopathologic entity. Subsequently, tumors with a similar histologic appearance, demonstrating the “rhabdoid” cells, have been found in a variety of extrarenal sites. METHODS: We report here a case of malignant rhabdoid tumor of the colon studied with selected molecular techniques. RESULTS AND CONCLUSIONS: This tumor demonstrated several unusual findings for malignant rhabdoid tumors of renal or extrarenal sites, including aneuploidy by flow cytometric analysis and a positive nuclear immunohistochemical staining for p53 protein, which suggests presence of p53 gene mutation. DNA analyses, however, failed to demonstrate the presence of point mutation in any of the ras family genes.     

In vivo tumor growth is inhibited by cytosolic iron deprivation caused by the expression of mitochondrial ferritin

Mitochondrial ferritin (MtFt) is a mitochondrial iron-storage protein whose function and regulation is largely unknown. Our previous results have shown that MtFt overexpression markedly affects intracellular iron homeostasis in mammalian cells. Using tumor xenografts, we examined the effects of MtFt overexpression on tumor iron metabolism and growth. The expression of MtFt dramatically reduced implanted tumor growth in nude mice. Mitochondrial iron deposition in MtFt-expressing tumors was directly observed by transmission electron microscopy. A cytosolic iron starvation phenotype in MtFt-expressing tumors was revealed by increased RNA-binding activity of iron regulatory proteins, and concomitantly both an increase in transferrin receptor levels and a decrease in cytosolic ferritin. MtFt overexpression also led to decreases in total cellular heme content and heme oxygenase-1 levels. In addition, elevated MtFt in tumors was also associated with a decrease in total aconitase activity and lower frataxin protein level. In conclusion, our study shows that high MtFt levels can significantly affect tumor iron homeostasis by shunting iron into mitochondria; iron scarcity resulted in partially deficient heme and iron-sulfur cluster synthesis. It is likely that deprivation of iron in the cytosol is the cause for the significant inhibition of xenograft tumor growth.     

Cell transformation and tumor induction by Abelson murine leukemia virus in the absence of helper virus.

We investigated the role of the Moloney helper virus, Moloney murine leukemia virus (Mo-MuLV), in cell transformation and tumor induction by the defective Abelson murine leukemia virus (Ab-MuLV). A molecular clone of Ab-MuLV (P160 strain) was transfected into the psi 2 packaging cell line, and helper virus-free Ab-MuLV (psi 2) was harvested from the supernatant medium. Ab-MuLV (psi 2) was as efficient as helper virus-containing Ab-MuLV (Mo-MuLV) in the transformation of primary bone marrow cells in vitro. Inoculation of weanling BALB/c mice with Ab-MuLV (psi 2) induced nonthymic pre-B-cell lymphomas with high efficiency and short latency (28 days). Adult BALB/c mice were less sensitive to tumor induction by a factor of 100. Ab-MuLV (psi 2) did not induce tumors in weanling C57BL/6 mice, unlike Ab-MuLV (Mo-MuLV). Examination of the proviral integration pattern in Ab-MuLV (psi 2)-induced tumor cell DNA revealed that each of the tumors contained a single integrated provirus. Immunoprecipitation of viral-encoded proteins in helper virus-free tumor cell lines detected the P160 Ab-MuLV-transforming protein; however, no trace of the gag, pol, and env helper virus-encoded proteins was found. Our results indicate that integration and expression of a single Ab-MuLV genome is sufficient for efficient transformation of primary bone marrow cells by Ab-MuLV in vitro and tumor induction in susceptible mice. However, the helper virus may contribute to tumor induction in weanling resistant mice.     

Dukes B colorectal cancer: distinct genetic categories and clinical outcome based on proximal or distal tumor location

PURPOSE: The aim of this study was to determine whether tumor location proximal or distal to the splenic flexure is associated with distinct molecular patterns and can predict clinical outcome in a homogeneous group of patients with Dukes B (T3-T4, N0, M0) colorectal cancer. It has been hypothesized that proximal and distal colorectal cancer may arise through different pathogenetic mechanisms. Although p53 and Ki-ras gene mutations occur frequently in distal tumors, another form of genomic instability associated with defective DNA mismatch repair has been predominantly identified in the proximal colon. To date, however, the clinical usefulness of these molecular characteristics remains unproven. METHODS: A total of 126 patients with a lymph node-negative sporadic colon or rectum adenocarcinoma were prospectively assessed with the endpoint of death by cancer. No patient received either radiotherapy or chemotherapy. p53 protein was studied by immunohistochemistry using DO-7 monoclonal antibody, and p53 and Ki-ras gene mutations were detected by single strand conformation polymorphism assay. RESULTS: During a mean follow-up of 67 months, the overall five-year survival was 70 percent. Nuclear p53 staining was found in 57 tumors (47 percent), and was more frequent in distal than in proximal tumors (55 vs. 21 percent; chi-squared test, P < 0.001). For the whole group, p53 protein expression correlated with poor survival in univariate and multivariate analysis (log-rank test, P = 0.01; hazard ratio = 2.16; 95 percent confidence interval = 1.12-4.11, P = 0.02). Distal colon tumors and rectal tumors exhibited similar molecular patterns and showed no difference in clinical outcome. In comparison with distal colorectal cancer, proximal tumors were found to be statistically significantly different on the following factors: mucinous content (P = 0.008), degree of histologic differentiation (P = 0.012), p53 protein expression, and gene mutation (P = 0.001 and 0.01 respectively). Finally, patients with proximal tumors had a marginally better survival than those with distal colon or rectal cancers (log-rank test, P = 0.045). CONCLUSION: In this series of Dukes B colorectal cancers, p53 protein expression was an independent factor for survival, which also correlated with tumor location. Eighty-six percent of p53-positive tumors were located in the distal colon and rectum. Distal colon and rectum tumors had similar molecular and clinical characteristics. In contrast, proximal neoplasms seem to represent a distinct entity, with specific histopathologic characteristics, molecular patterns, and clinical outcome. Location of the neoplasm in reference to the splenic flexure should be considered before group stratification in future trials of adjuvant chemotherapy in patients with Dukes B tumors.     

A case of progressing focal nodular hyperplasia and its molecular expression pattern

We report the case of an adult male with progressing focal nodular hyperplasia (FNH). Although imaging studies suggested that the tumor was a classical FNH, the tumor biopsy showed glutamine synthetase expression and heat shock protein 70 in part of the tumor. As we could not definitely distinguish this case of FNH from early hepatocellular carcinoma (HCC), we performed laparoscopic partial hepatectomy. The surgical resected specimen showed that the tumor had a central scar with vascular and cholangiolar proliferation, which is compatible with FNH. Immunohistochemical analysis showed that the molecular expression pattern was compatible with FNH in the center of the tumor, whereas it partly resembled early HCC in the periphery of the tumor. FNH progression is occasionally found, and the molecular pattern of the progressing area in FNH might resemble that of early HCC due to morphologic and phenotypic changes induced by the regenerative mechanism and the alteration of blood flow. We should carefully observe progressing FNH.     

Keratin synthesis in normal mouse epithelia and in squamous cell carcinomas: evidence in tumors for masked mRNA species coding for high molecular weight keratin polypeptides.

Transplantable mouse squamous cell carcinomas (SCC), originally derived either from back skin or forestomach epithelium, do not synthesize high molecular weight keratin polypeptides [greater than 60 kilodaltons (kDa)] involved in the process of terminal differentiation in the corresponding normal tissues. The in vivo tumor keratin spectra consist of only low molecular weight keratin subunits at 60, 58, 52, 50, 47, and 46 kDa, each encoded by its own mRNA and encountered also in normal epidermis and forestomach epithelium. In addition, both tumors express a mRNA-dependent 40-kDa protein, whereas a 56-kDa protein and its mRNA are selectively found only in the forestomach tumor. Translation of mRNAs from both tumors in a cell-free system does not only generate analogues of the in vivo tumor keratin polypeptides, but also both SCC possess an additional mRNA coding in vitro for a 67-kDa keratin subunit that is not expressed, however, in the carcinomas in vivo. The identity of this in vitro synthesized keratin member with a 67-kDa keratin polypeptide of both normal epidermis and forestomach epithelium was confirmed by comparison of charge properties and peptide mapping. With regard to this particular keratin polypeptide, the tumors are obviously able to sequester the polypeptide's mRNA in an untranslatable state in the cells.     

On the role of protein synthesis in the response of adrenal tumor cells to ACTH.

Y-1 adrenal tumor cells were incubated with aminoglutethimide with and without ACTH. Greater production of pregnenolone from endogenous cholesterol was observed (after washing to remove aminoglutethimide) in mitochondria from cells incubated with aminoglutethimide and ACTH than in those from cells incubated with aminoglutethimide alone. This response was inhibited by cycloheximide and puromycin but not by chloramphenicol or actinomycin D. ACTH increased the incorporation of [3H]tyrosine into protein associated with mitochondria but not into total cell protein or protein of postmitochondrial supernatant. This response did not require aminoglutethemide block and was inhibited by cycloheximide and puromycin but not by chloramphenicol or actinomycin D. Dibutyryl cyclic AMP produced both of these responses (increased production of pregnenolone and synthesis of protein associated with mitochondria). The concentration of cycloheximide required to cause 50% inhibition of the responses to ACTH and dibutyryl cyclic AMP was approximately the same for steroidogenesis by whole cells, for production of pregnenolone by isolated mitochondria, for incorporation of [3H]tyrosine into Y-1 cell protein and for the increase in synthesis of protein associated with mitochondria produced by ACTH (0.08--0.2 microgram/ml). Disc gel electrophoresis revealed that the increased incorporation of [3H]tyrosine involved two proteins corresponding to molecular weight of approximately 27,000 and 13,000 respectively. These observations suggest that ACTH promotes synthesis of protein(s) by cytoplasmic ribosomes on stable messenger RNA, that the protein(s) becomes associated with mitochondria and that the protein(s) includes one or more which are associated with the increase in production of pregnenolone produced in mitochondria by the addition of ACTH to adrenal cells.     

A metastatic case of gastrointestinal stromal tumor (GIST) treated with imatinib mesylate (Glivec(®)) in Mali

Gastrointestinal stromal tumors (GIST) usually showing a spindle cells pattern of cell proliferation have recently benefit from a molecular definition. Indeed imatinib mesylate (Gleevec(?)) treatment has dramatically improved the management of these tumors as they frequently express the c-kit oncogene. We report the first case of a metastatic gastric GIST in a man of 45 years diagnosed and treated in Mali. The gastric tumor was particularly aggressive with a large intra-abdominal and mesenteric spreading and liver metastases. The diagnosis was done on the CD117 and CD34 expression in the tumor sample obtained by laparotomy. After a 34 months 400mg/day imatinib mesylate (Gleevec(?)) treatment a dramatic tumor regression was obtained.     

A primary solitary tumor of the lesser omentum with immunohistochemical features of gastrointestinal stromal tumors

We here present a primary solitary tumor of the lesser omentum that was found in a 71-yr-old woman. Differential diagnosis could not be made preoperatively; therefore, histopathological examination including immunohistochemical studies were performed to determine the nature of the tumor.
The resected specimen, measuring 17 cm at the largest point, consisted of the outer solid part and the inner multiloculated cysts. Microscopically, the tumor was characterized by interlacing bundles of elongated spindle cells, with the nuclei focally showing a palisading pattern. However, skeinoid fibers were not observed anywhere. One to three mitoses per 50 high power fields were observed. Immunohistochemically, the tumor was negative for S-100 protein and smooth muscle–specific actin, but stained positive for CD34. The microscopic features were consistent with those of potentially malignant gastrointestinal stromal tumors.
Stromal tumors that represent the differentiation toward neither typical leiomyomas or schwannomas rarely occur in the lesser omentum with only one such instance having been reported to date. Due to this rarity, it is difficult to make the differential diagnosis preoperatively, even with existing imaging techniques, and predicting the clinical behavior of such omental tumors is also often difficult. Therefore, complete resection should be performed when such tumors are encountered in daily practice.     

Histopathological studies of mucin-producing carcinoma of the bile duct

Mucin-producing carcinoma (MPC) of the bile duct produces large amounts of mucin. As many aspects of the characteristic biological pattern of invasion and origin of this tumor are unclear, we investigated its pathological molecular biology and association with peribiliary glands. Molecular biologically, MPC with multiple tumors had a higher tumor proliferation potency than MPC consisting of a single tumor. Even multiple tumors with high malignant potential showed little evidence of lymphatic invasion, and there was little venous or perineural invasion. Findings in regard to the peribiliary glands (PGs) suggested that PGs are involved in the origin and extension of MPC. Mucinous PGs under the main tumor were exhibited beneath the dysplasia and non-neoplastic epithelium, whereas mucinous PGs under MPC with multiple tumors contained neoplastic cells. PGs secreted large quantities of mucin. We conclude that neoplastic cells in PGs caused cell proliferation toward the bile duct lumen.     

A chemical genomics screen highlights the essential role of mitochondria in HIF-1 regulation

Hypoxia-inducible factor-1 (HIF-1) plays an essential role in tumor development and progression by regulating genes that are vital for proliferation, glycolysis, angiogenesis, and metastasis. To identify strategies of targeting the HIF-1 pathway, we screened a siRNA library against the entire druggable genome and a small-molecule library consisting of 691,200 compounds using a HIF-1 reporter cell line. Although the siRNA library screen failed to reveal any druggable targets, the small-molecule library screen identified a class of alkyliminophenylacetate compounds that inhibit hypoxia-induced HIF-1 reporter activity at single-digit nanomolar concentrations. These compounds were found to inhibit hypoxia but not deferoxamine-induced HIF-1alpha protein stabilization. Further analysis indicated that the alkyliminophenylacetate compounds likely inhibit the HIF-1 pathway through blocking the hypoxia-induced mitochondrial reactive oxygen species (ROS) production. Strikingly, all of the nonalkyliminophenylacetate HIF-1 inhibitors identified from the small-molecule library screen were also found to target mitochondria like the alkyliminophenylacetate compounds. The exclusive enrichment of mitochondria inhibitors from a library of >600,000 diverse compounds by using the HIF-1 reporter assay highlights the essential role of mitochondria in HIF-1 regulation. These results also suggest that targeting mitochondrial ROS production might be a highly effective way of blocking HIF-1 activity in tumors.     

Variable beta-catenin expression in colorectal cancers indicates tumor progression driven by the tumor environment

Invasion and dissemination of well-differentiated carcinomas are often associated with loss of epithelial differentiation and gain of mesenchyme-like capabilities of the tumor cells at the invasive front. However, when comparing central areas of primary colorectal carcinomas and corresponding metastases, we again found the same differentiated epithelial growth patterns. These characteristic phenotypic changes were associated with distinct expression patterns of beta-catenin, the main oncogenic protein in colorectal carcinomas, and E-cadherin. Nuclear beta-catenin was found in dedifferentiated mesenchyme-like tumor cells at the invasive front, but strikingly, as in central areas of the primary tumors, was localized to the membrane and cytoplasm in polarized epithelial tumor cells in the metastases. This expression pattern was accompanied by changes in E-cadherin expression and proliferative activity. On the basis of these data, we postulate that an important driving force for progression of well-differentiated colorectal carcinomas is the specific environment, initiating two transient phenotypic transition processes by modulating intracellular beta-catenin distribution in tumor cells.     

结直肠癌外周血肿瘤标志物的研究进展

结直肠癌是临床上常见的恶性肿瘤之一,如能早期发现、早期治疗,则有可能治愈.随着分子生物学的发展,外周血中检测肿瘤标志物在结直肠癌筛查中的应用,结直肠癌的早期发现成为可能.然而,肿瘤与其标志物之间的关系错综复杂,这就需要我们寻找相关的肿瘤标志物来进行检测.本文就常用及新发现的外周血肿瘤标志物综述如下.     

女性肺癌组织中Ki 67、P53表达与临床的相关性研究

目的观察女性肺癌组织中Ki67和p53表达水平与临床的关系。方法统计手术切除的女性肺癌标本,应用免疫组织化学方法检测肺癌组织中Ki67、p53的表达水平,并随访不同表达水平的Ki67、p53对预后的影响。结果 Ki67蛋白阳性率为76.79%,P53蛋白阳性率为73.21%,Ki67蛋白表达在女性肺癌不同病理类型、肿瘤分期、有无淋巴结转移上有显著性差异(P0.05),分期晚、淋巴结转移、非腺癌组表现出高Ki67表达,而本研究发现P53表达在年龄、分期、淋巴结有无转移、女性肺癌病理类型上均无统计学差异。结论 Ki67蛋白表达对临床女性肺癌诊断和预后判断可提供有效的分子生物学依据,Ki67蛋白阳性者预后差。     

Size heterogeneity of beta-MSH in ectopic ACTH-producing tumors: presence of beta-LPH-like peptide.

Gel chromatographic, immunologic and biologic properties of beta-melanocyte-stimulating hormone (beta-MSH) in tumor tissues obtained from eight patients with the ectopic ACTH syndrome were studied and compared to those of pituitary beta-MSH. Size heterogeneity of immunoreactive beta-MSH was found in all the tumors studied as well as in normal human pituitaries. Both the tumors and pituitaries contained immunoreactive beta-MSH of a larger molecular size than the well-characterized beta-MSH of small molecular size. The large molecular weight beta-MSH also predominated in the plasma. It was found to be bioactive by an in vitro MSH assay, immunologically indistinguishable from human beta-MSH, and chromatographically very similar to beta-lipotropic hormone (beta-LPH). Tryptic digestion of the large molecular weight beta-MSH under controlled conditions promptly produced bioactive beta-MSH of small molecular size, followed by the appearance of immunologically active but biologically inert fragments. These results suggest that the ectopic ACTH-producing tumor as well as the pituitary elaborate beta-LPH-like peptide which might be the predominant component of immunoreactive beta-MSH in man.     

Dukes B colorectal cancer

PURPOSE: The aim of this study was to determine whether tumor location proximal or distal to the splenic flexure is associated with distinct molecular patterns and can predict clinical outcome in a homogeneous group of patients with Dukes B (T3–T4, N0, M0) colorectal cancer. It has been hypothesized that proximal and distal colorectal cancer may arise through different pathogenetic mechanisms. Althoughp53 and Ki-ras gene mutations occur frequently in distal tumors, another form of genomic instability associated with defective DNA mismatch repair has been predominantly identified in the proximal colon. To date, however, the clinical usefulness of these molecular characteristics remains unproven. METHODS: A total of 126 patients with a lymph node-negative sporadic colon or rectum adenocarcinoma were prospectively assessed with the endpoint of death by cancer. No patient received either radiotherapy or chemotherapy. p53 protein was studied by immunohistochemistry using DO-7 monoclonal antibody, andp53 and Ki-ras gene mutations were detected by single strand conformation polymorphism assay. RESULTS: During a mean follow-up of 67 months, the overall five-year survival was 70 percent. Nuclearp53 staining was found in 57 tumors (47 percent), and was more frequent in distal than in proximal tumors (55vs. 21 percent; chi-squared test,P<0.001). For the whole group, p53 protein expression correlated with poor survival in univariate and multivariate analysis (log-rank test,P=0.01; hazard ratio=2.16; 95 percent confidence interval=1.12–4.11,P=0.02). Distal colon tumors and rectal tumors exhibited similar molecular patterns and showed no difference in clinical outcome. In comparison with distal colorectal cancer, proximal tumors were found to be statistically significantly different on the following factors: mucinous content (P=0.008), degree of histologic differentiation (P=0.012), p53 protein expression, and gene mutation (P=0.001 and 0.01 respectively). Finally, patients with proximal tumors had a marginally better survival than those with distal colon or rectal cancers (log-rank test,P=0.045). CONCLUSION: In this series of Dukes B colorectal cancers, p53 protein expression was an independent factor for survival, which also correlated with tumor location. Eighty-six percent ofp53-positive tumors were located in the distal colon and rectum. Distal colon and rectum tumors had similar molecular and clinical characteristics. In contrast, proximal neoplasms seem to represent a distinct entity, with specific histopathologic characteristics, molecular patterns, and clinical outcome. Location of the neoplasm in reference to the splenic flexure should be considered before group stratification in future trials of adjuvant chemotherapy in patients with Dukes B tumors.Supported by a grant from the SICPA foundation and by the Swiss National Science FoundationRead at the meeting of The American Society of Colon and Rectal Surgeons, Boston, Massachusetts, June 24 to 29, 2000.     

p53 Mutation Pattern and Expression of c-erbB2 and c-met in Gastric Cancer: Relation to Histological Subtypes, Helicobacter pylori Infection, and Prognosis

The molecular mechanisms of Helicobacter pylori associated tumor development are poorly understood. The spectra of genetic alterations in neoplasms may provide clues to the molecular carcinogenesis of a tumor and may be relevant for the prognosis of the patients. We investigated the p53 mutation pattern and the protein expression of p53, c-erbB2, and c-met in 42 gastric cancers and correlated these alterations with H. pylori infection, histological subtypes and survival of the patients after curative resection. There were no differences in the incidences of the expression of p53, c-erbB2, and c-met in the tumor tissues according to H. pylori infection. Fifteen p53 mutations in 12 (29%) tumors were identified. More p53 mutations were found in patients with positive serology for H. pylori (43% vs 14%). This difference was not significant, but the small sample size may be insufficient to detect a potential statistical difference. There was neither a H. pylori-associated p53 hot-spot codon mutation nor a H. pylori characteristic mutational pattern of p53. Positive lymph nodes (P = 0.0061) and p53 mutations (P = 0.0035) were the only significant bad prognostic markers for survival after curative resection of the gastric cancers in our study. Our study does not indicate a unique molecular mechanism of p53 mutagenesis through H. pylori infection. The fact that p53 mutations were significantly correlated with poor survival of patients after potentially curative resection of gastric cancer may have clinical implications for multimodal therapies.