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Numerous esophageal squamous cell carcinoma (ESCC) patients exhibit tumor recurrence following radical resection. Invasion and metastasis are key factors in poor prognosis following esophagectomy. In the present study, two‑dimensional gel electrophoresis (2-DE) and matrix‑assisted laser desorption/ionization time-of-flight mass spectrometry were used to define patterns of protein expression in ESCC tissues at different pathological stages. The expression levels of identified proteins were determined by immunohistochemistry and western blotting. A total of fifteen protein spots with >2-fold differences were observed when comparing results of 2-DE for stage III and stage I ESCC tissue sample. A total of 12 proteins were identified by mass spectrometry analysis and database searches. The results of immunohistochemistry and western blotting demonstrated expression levels of tropomyosin 3 (TPM3) were higher in stage III ESCC tissue compared with stage I (P

According to existing reports, mutations in the slow tropomyosin gene (TPM3) may lead to congenital fiber-type disproportion (CFTD), nemaline myopathy (NM) and cap myopathy (CD). They are all congenital myopathies and are associated with clinical, pathological and genetic heterogeneity. A ten-year-old girl with scoliosis was unable to wean from mechanical ventilation after total intravenous anesthesia. The girl has scoliosis, respiratory insufficiency, motion delay and muscle weakness; her younger brother has a similar physiology but does not have scoliosis or respiratory insufficiency, and her parents are healthy. We conducted genetic testing and found a c.502C > G (p.R168G) heterozygous mutation in the family. This mutation originated from the father and was autosomal dominant. Muscle biopsy results indicated that no special structures were present, and the type I fiber ratio was not notably high compared to previous reports. Although the family members have the same mutations, their clinical manifestations are quite different.

Abnormal expression of microRNA (miR) is associated with the occurrence and progression of various types of cancers, including papillary thyroid carcinoma (PTC). In the present study, the aim was to explore miR-486-5p expression and its role in PTC, as well as to investigate the biological function of its potential target genes. The expression levels of miR-486-5p and its clinicopathological significance were examined in 507 PTC and 59 normal thyroid samples via The Cancer Genome Atlas (TCGA). Subsequently, the results were validated using data from Gene Expression Omnibus (GEO) and ArrayExpress. Receiver operating characteristic and summary receiver operating characteristic curves were used to assess the ability of miR-486-5p in distinguishing PTC from normal tissue. Furthermore, potential miR-486-5p mRNA targets were identified using 12 prediction tools and enrichment analysis was performed on the encoding genes using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. The expression levels of miR-486-5p were consistently downregulated in PTC compared with in normal tissue across datasets from TCGA, GEO ({"type":"entrez-geo","attrs":{"text":"GSE40807","term_id":"40807"}}GSE40807, {"type":"entrez-geo","attrs":{"text":"GSE62054","term_id":"62054"}}GSE62054 and {"type":"entrez-geo","attrs":{"text":"GSE73182","term_id":"73182"}}GSE73182) and ArrayExpress (E-MTAB-736). The results also demonstrated that miR-486-5p expression was associated with cancer stage (P=0.003), pathologic lymph node (P=0.047), metastasis (P=0.042), neoplasm (P=0.012) and recurrence (P=0.016) in patients with PTC. In addition, low expression of miR-486-5p in patients with PTC was associated with a worse overall survival. A total of 80 miR-486-5p-related genes were observed from at least 9 of 12 prediction platforms, and these were involved in ‘hsa05200: Pathways in cancer’ and ‘hsa05206: MicroRNAs in cancer’. Finally, three hub genes, CRK like proto-oncogene, phosphatase and tensin homolog and tropomyosin 3, were identified as important candidates in tumorigenesis and progression of PTC. In conclusion, it may be hypothesized that miR-486-5p contributes towards PTC onset and progression, and may act as a clinical target. However, in vitro and in vivo experiments are required to validate the findings of the present study.

Inflammatory myofibroblastic tumor (IMT) is a mesenchymal tumor that can arise from anywhere in the body. Anaplastic lymphoma kinase (ALK) gene rearrangements, most often resulting in the tropomyosin 3 (TPM3)-ALK fusion gene, are the main causes of IMT. However, the mechanism of malignant transformation in IMT has yet to be elucidated. The purpose of this study was to clarify the role of the TPM3 region in the transformation of IMT via TPM3-ALK. Lentivirus vectors containing a TPM3-ALK fusion gene lacking various lengths of TPM3 were constructed and expressed in HEK293T and NIH3T3 cell lines. Focus formation assay revealed loss of contact inhibition in NIH3T3 cells transfected with full-length TPM3-ALK, but not with ALK alone. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) revealed that TPM3-ALK dimerization increased in proportion to the length of TPM3. Western blot showed phosphorylation of ALK, ERK1/2, and STAT3 in HEK293T cells transfected with TPM3-ALK. Thus, the coiled-coil structure of TPM3 contributes to the transforming ability of the TPM3-ALK fusion protein, and longer TPM3 region leads to higher dimer formation.

The burden of colorectal cancer (CRC) is considerable&mdash, approximately 1.8 million people are diagnosed each year with CRC and of these about half will succumb to the disease. In the case of CRC, there is strong evidence that an early diagnosis leads to a better prognosis, with metastatic CRC having a 5-year survival that is only slightly greater than 10% compared with up to 90% for stage I CRC. Clearly, biomarkers for the early detection of CRC would have a major clinical impact. We implemented a coherent gel-based proteomics biomarker discovery platform for the identification of clinically useful biomarkers for the early detection of CRC. Potential protein biomarkers were identified by a 2D gel-based analysis of a cohort composed of 128 CRC and site-matched normal tissue biopsies. Potential biomarkers were prioritized and assays to quantitatively measure plasma expression of the candidate biomarkers were developed. Those biomarkers that fulfilled the preset criteria for technical validity were validated in a case-control set of plasma samples, including 70 patients with CRC, adenomas, or non-cancer diseases and healthy individuals in each group. We identified 63 consistently upregulated polypeptides (factor of four-fold or more) in our proteomics analysis. We selected 10 out of these 63 upregulated polypeptides, and established assays to measure the concentration of each one of the ten biomarkers in plasma samples. Biomarker levels were analyzed in plasma samples from healthy individuals, individuals with adenomas, CRC patients, and patients with non-cancer diseases and we identified one protein, tropomyosin 3 (Tpm3) that could discriminate CRC at a significant level (p = 0.0146). Our results suggest that at least one of the identified proteins, Tpm3, could be used as a biomarker in the early detection of CRC, and further studies should provide unequivocal evidence for the real-life clinical validity and usefulness of Tpm3.

Background and Objectives: The heat-shock response modulates contractility of vascular smooth muscles. With complementary deoxyribonucleic acid microarray, we tried to identify the novel genes that are involved in the regulation of vascular contraction after heat shock. Materials and Methods: Human radial artery strips were mounted in organ baths, exposed at 42°C for 45 minutes, and returned to equilibrate at 37°C. This study examined gene expression profile associated with heat-shock response in radial arteries of patients with hyperlipidemia by using a microarray that contained 5763 human cDNA. The results of microarray hybridization experiments from the radial arteries of 4 different subjects were analyzed and classified by the cluster program. Results: Among these differentially-expressed genes, Hsp70, Hsp10, αB-crystallin, and Hsp60 were significantly increased by the heat shock response. Of non-HSP genes, 15 genes increased, while 22 genes decreased. Among these 37 genes, αB-crystallin (CRYAB) (up 1.92fold), myosin, light polypeptide kinase transcript variant 8, 6 (up 1.70-fold, up 1.68-fold), catenin (cadherin-associated protein, alpha-like 1) (down-0.57 fold) and tropomyosin 3 (down 0.68-fold) were thought to be related with the contraction. Real-time quantitative polymerase chain reaction showed that Hsp70, Hsp10 and αB-crystallin were significantly increased. Conclusion: Gene expression profile by heat shock provides information about genes implicated in augmentation of vascular contraction after heat shock. (Korean Circ J 2012;42:479-486)

To explore the potential tumor markers for lung adenocarcinoma, two-dimensional gel electrophoresis (2DE) coupled with nano-LC/MS/MS was used to analyze the differentially expressed proteins in 10 surgical resected lung adenocarcinoma tissues. 16 proteins were significantly different between the cancer tissue and adjacent normal tissue. Galectin-1, peroxiredoxin II (Prx II), proapolipoprotein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase A, enolase 1, neuropolypeptide h3, Prx V, cyclophilin A, vimentin, protein disulfide isomerase (PDI), tropomyosin 3 (TPM 3), glutathione S-transferase Pi (GST-Pi), manganese superoxide dismutase (MnSOD), and cofilin 1 were up-regulated in the cancer tissue. On the other hand, profilin was down-regulated in the cancer tissue. Among these proteins, six proteins were validated by Western blot analysis. The identified proteins contributing to the spectrum of cancer progression may be used as potential diagnostic biomarkers for lung adenocarcinomas.

Through comparative gene mapping, NICE-3, which is closely linked to tropomyosin 3 in human chromosome 1, was selected to be investigated as a new candidate gene associated with the muscle development in pigs. This gene was sequenced, chromosome mapped, expression analyzed, subcellularly localized, and promoter activity analyzed. After screening and sequencing, porcine NICE-3 was found in a bacterial artificial chromosome clone containing tropomyosin 3. Quantitative reverse transcription-polymerase chain reaction revealed that NICE-3 mRNA was widely expressed, with highest expression levels in longissimus dorsi muscles, followed by heart, biceps femoris, liver, kidney, back fat, and lowest expression levels in spleen, brain, lymph, lung, stomach, and small and large intestines. Fluorescence and confocal microscopy assay demonstrated that the fusion protein, GFP-NICE-3, was distributed throughout the cytoplasm, including the plasma membrane. NICE-3 was mapped to Sus scrofa chromosome 4, in a region of conserved synteny with human chromosome 1, where the homologous human gene is localized. Results of dual reporter gene assays and mutation experiments combined with electrophoresis mobility shift assays showed that the retinoid X receptor might be an important transcription factor affecting the promoter activity of this gene.

Purpose Recent evidence shows that nitric oxide (NO) may exhibit both pro-cancer and anti-cancer activities. The present study aimed to determine the differentially expressed proteins in NO-treated NIH/3T3 fibroblasts in order to investigate whether NO induces proteins with pro-cancer or anti-cancer effects. Materials and methods The cells were treated with 300 μM of an NO donor 3,3-bis-(aminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC-18) for 12 h. The changed protein patterns, which were separated by two-dimensional electrophoresis using pH gradients of 4-7, were conclusively identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the peptide digests. Results Seventeen differentially expressed proteins were identified in NOC-18-treated cells. Nine proteins [vinculin protein, keratin 19, ubiquitous tropomodulin, F-actin capping protein (α1 subunit), tropomyosin 3, 26S proteasome-associated pad1 homolog, T-complex protein 1 (e subunit) N(G)-dimethylarginine dimethylaminohydrolase, and heat shock protein 90] were increased and eight proteins (heat shock protein 70, glucosidase II, lamin B1, calreticulin, nucleophosmin 1, microtubule-associated protein retinitis pigmentosa/end binding family member 1, 150 kD oxygen-regulated protein precursor, and heat shock 70-related protein albino or pale green 2) were decreased by NOC-18 in the cells. Thirteen proteins are related to the suppression of cancer cell proliferation, invasion, and metastasis while two proteins (heat shock protein 90 and N(G)-dimethylarginine dimethylaminohydrolase) are related to carcinogenesis. The functions of 150 kD oxygen-regulated protein precursor and T-complex protein 1 (e subunit) are unknown in relation to carcinogenesis. Conclusion Most proteins differentially expressed by NOC-18 are involved in inhibiting cancer development.

Fresh, superficial transitional cell carcinomas (TCCs) of low-grade atypia (3 grade I, Ta; 6 grade II, Ta), as well as primary cultures derived from them were labeled with [35S]methionine for 16 h, between 2 and 6 days after inoculation. Whole protein extracts were subjected to IEF (isoelectric focusing) two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) followed by autoradiography. Proteins were identified by a combination of proteomic technologies that included microsequencing, mass spectrometry, 2-D PAGE immunoblotting and comparison with the bladder TCC protein database available on the internet (http://biobase.dk/cgi-bin/celis). Comparison of the IEF 2-D gel protein profiles of fresh tumors and their primary cultures showed that the overall expression profiles were strikingly similar, although differing significantly in the levels of several proteins whose rate of synthesis was differentially regulated in at least 85% of the tumor/culture pairs as a result of the short-term culturing. Most of the proteins affected by culturing were upregulated and among them we identified components of the cytoskeleton (keratin 18, gelsolin and tropomyosin 3), a molecular chaperone (hsp 28), aldose reductase, GST pi, metastasin, synuclein, the calreticulin precursor and three polypeptides of unknown identity. Only four major proteins were downregulated, and these included two fatty acid-binding proteins (FABP:FABP5 and A-FABP) which are thought to play a role in growth control, the differentiation-associated keratin 20, and the calcium-binding protein annexin V. Proteins that were differentially regulated in only some of the cultured tumors included alpha-enolase, triosphosphate isomerase, members of the 14-3-3 family, hnRNPs F and H, PGDH, hsp (heat-shock protein) 60, BIP, the interleukin-1 receptor antagonist, the nucleolar protein B23, as well as several proteins of yet unknown identity. The suitability of in vitro bladder tumor culture models to study complex biological phenomena such as malignancy and invasion is discussed.

Although the highest incidence of esophageal squamous cell carcinoma (ESCC) has repeatedly been reported from Persia (Iran), nevertheless the so far proteomic published reports were limited to one study on tissue specimens. Here we report the proteome of a newly established cell line from Persian ESCC patients and compare it with the normal primary cell proteome. Among polypeptides, whose expression was different in cell line sixteen polypeptides were identified by MALDI/TOF/TOF spectrometry. S100-A8 protein, annexin A1, annexin A2, regulatory subunit of calpain, subunit alpha type-3 of proteasome and glutamate dehydrogenase 1 were proteins down-regulated in cell line while peroxiredoxin-5, non-muscle myosin light polypeptide 6, keratin 1, annexin A4, keratin 8, tropomyosin 3, stress-induced-phosphoprotein 1 and albumin were found to be subject of up-regulation in cell line compared to the primary normal cells. The proteomic results were further verified by western blotting and RT-PCR on annexin A1 and keratin 8. In addition, among the aforementioned proteins, glutamate dehydrogenase 1, regulatory subunit of calpain, subunit alpha of type-3 proteasome and annexin A4 are proteins whose deregulation in ESCC is reported for the first time by this study.

Cisplatin is used widely for treatment of a variety of cancer diseases. Recently, however, the use of cisplatin is restricted because of its adverse effects such as hepatotoxicity. There is no study with current proteomics technology to evaluate cisplatin-induced hepatotoxicity, even if some studies have reported on the hepatotoxicity. In this study, proteomic as well as genomic analyses have been used for identification of proteins and genes that respond to cisplatin treatment in rat primary hepatocytes. To investigate the hepatotoxic effects of cisplatin, rat primary hepatocytes were treated with an IC(20) concentration for 24 h. From proteomic analysis based on label-free quantitation strategy, cisplatin induced 76 up-regulated and 19 down-regulated proteins among 325 distinct proteins. In the mRNA level, genomic analysis revealed 72 up-regulated and 385 down-regulated genes in the cisplatin-treated group. Based on these two analyses, 19 pathways were commonly altered, whereas seven pathways were identified only by proteomic analysis, and 19 pathways were identified only by genomic analysis. Overall, this study explained the mechanism of cisplatin-induced hepatotoxicity with two points of view: well known pathways including drug metabolism, fatty acid metabolism, and glycolysis/TCA cycle and little known pathways including urea cycle and inflammation metabolism, for hepatotoxicity of other toxic agents. Up-regulated proteins detected by proteomic analysis in the cisplatin-treated group: FBP1 (fructose 1,6-bisphosphatase 1), FASN (fatty acid synthase), CAT (catalase), PRDX1 (peroxiredoxin-1), HSPD1 (60-kDa heat shock protein), MDH2 (malate dehydrogenase 2), and ARG1 (arginase 1), and also down-regulated proteins in the cisplatin-treated group: TPM1 (tropomyosin 1), TPM3 (tropomyosin 3), and CTSB (cathepsin B), were confirmed by Western blot analysis. In addition, up-regulated mRNAs detected by microarray analysis in the cisplatin-treated group: GSTA2, GSTT2, YC2, TXNRD1, CYP2E1, CYP2C13, CYP2D1, ALDH17, ARG1, ARG2, and IL-6, and also down-regulated mRNAs: CYP2C12, CYP26B1, TPM1, and TPM3, were confirmed by RT-PCR analysis. In case of PRDX1, FASN, and ARG1, they were further confirmed by immunofluorescence analysis. Through the integrated proteomic and genomic approaches, the present study provides the first pathway map related to cisplatin-induced hepatotoxicity, which may provide new insight into the mechanism of hepatotoxicity.

Nemaline myopathy, the most common non-dystrophic congenital myopathy, is caused by mutations in six genes, all of which encode thin-filament proteins, including NEB (nebulin) and TPM3 (α tropomyosin). In contrast to the mechanisms underlying weakness in NEB-based myopathy, which are related to loss of thin-filament functions normally exerted by nebulin, the pathogenesis of muscle weakness in patients with TPM3 mutations remains largely unknown. Here, we tested the hypothesis that the contractile phenotype of TPM3-based myopathy is different from that of NEB-based myopathy and that this phenotype is a direct consequence of the loss of the specific functions normally exerted by tropomyosin. To test this hypothesis, we used a multidisciplinary approach, including muscle fiber mechanics and confocal and electron microscopy to characterize the structural and functional phenotype of muscle fibers from five patients with TPM3-based myopathy and compared this with that of unaffected control subjects. Our findings demonstrate that patients with TPM3-based myopathy display a contractile phenotype that is very distinct from that of patients with NEB-based myopathy. Whereas both show severe myofilament-based muscle weakness, the contractile dysfunction in TPM3-based myopathy is largely explained by changes in cross-bridge cycling kinetics, but not by the dysregulation of sarcomeric thin-filament length that plays a prominent role in NEB-based myopathy. Interestingly, the loss of force-generating capacity in TPM3-based myopathy appears to be compensated by enhanced thin-filament activation. These findings provide a scientific basis for differential therapeutics aimed at restoring contractile performance in patients with TPM3-based versus NEB-based myopathy.

Choroid plexus homogenates from 27 cases with Alzheimer disease-related pathology (AD), stages I/0 (n = 5), III-IV/0-B (n = 15) and V-VI/B-C (n = 7) and 3 age-matched controls (no clinical symptoms, no neuropathological lesions) were processed for gel electrophoresis and western blotting for oxidation markers carboxymethyl-lysine (CML) and N-carboxyethyl-lysine (CEL). Increased CEL and CML expression was seen in AD cases stages IVB, and V-VI/B-C when compared to controls and cases with AD-related pathology classified as I/0 and III/0. Variable stress damage was seen in stage III/B. Although lower stages of AD did not show beta-amyloid deposition in the choroid plexus, the amount of beta-amyloid was very variable at stages V/VI as revealed by western blotting, suggesting that other factors in addition to local fibrillar beta-amyloid were associated with oxidative damage in the choroid plexus. Two-dimensional gel electrophoresis and western blotting to CEL and CML in combination with mass spectrometry disclosed increased intensity of variable spots in AD cases leading to the identification of tyrosine 3/tryptophan 5-monooxygenase activation protein, zeta polypeptide, tropomyosin 3 isoform, and apolipoprotein A-II (ApoA-II) as targets of increased oxidative damage in AD. Oxidation of these proteins may result in impaired protein interactions, protein folding and protein kinase activity; abnormal function of endothelial and vascular smooth muscle cells; and impaired HDL-cholesterol metabolism in the choroid plexus in advanced stages of AD.

Heroin use is postulated to act as a cofactor in the neuropathogenesis of human immunodeficiency virus (HIV-1) infection. Astrocytes, integral components of the CNS, are reported to be susceptible to HIV-1 infection. Upon activation, astrocytes release a number of immunoregulatory products or modulate the expression of a number of proteins that foster the immunopathogenesis of HIV-1 infection. However, the role of heroin on HIV-1 infectivity and the expression of the proteome of normal human astrocytes (NHA) have not been elucidated. We hypothesize that heroin modulates the expression of a number of proteins by NHA that foster the neuoropathogenesis of HIV-1 infection. We utilized LTR amplification and the p24 antigen assay to quantitate the effect of heroin on HIV-1 infectivity while difference gel electrophoresis (DIGE) combined with protein identification through high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to analyze the effects of heroin on the proteomic profile of NHA. Results demonstrate that heroin potentiates HIV-1 replication in NHA. Furthermore, heroin significantly increased protein expression levels for protein kinase C (PKC), reticulocalbin 1 precursor, reticulocalbin 1, tyrosine 3-monooxgenase/tryptophan 5-monooxgenase activation protein, chloride intracellular channel 1, cathepsin D preproprotein, galectin 1 and myosin light chain alkali. Heroin also significantly decreased protein expression for proliferating cell nuclear antigen, proteasome beta 6 subunit, tropomyosin 3, laminin receptor 1, tubulin alpha 6, vimentin, EF hand domain family member D2, Tumor protein D54 (hD54), ATP synthase, H+ transporting, mitochondrial F1 complex and ribosomal protein S14. Identification of unique, heroin-induced proteins may help to develop novel markers for diagnostic, preventative and therapeutic targeting in heroin using subjects.

Tuberous sclerosis (TSC) is an autosomal dominant tumour suppressor gene syndrome affecting about 1 in 6000 individuals. It is characterized by mental retardation and epilepsy. A variety of tumours characteristically occur in different organs of TSC patients. Typically, highly epileptogenic dysplastic lesions (tubers) composed of abnormal shaped neurones can be detected in the cerebral cortex. Two tumour suppressor genes have been shown to be responsible for this disease: TSC1, encoding hamartin, and TSC2, encoding tuberin. In this study we performed a proteomic approach of two-dimensional gel electrophoresis with subsequent mass spectrometrical identification of protein spots after ectopic overexpression of human TSC1 or TSC2. We found the protein levels of α1-tubulin, protein disulfide isomerase, tropomyosin 3 and 5 and vimentin to be regulated by the two tuberous sclerosis gene products. The here presented findings suggest that deregulation of the control of these target proteins might contribute to the development of tumours in tuberous sclerosis patients. These data provide important new insights into the molecular development of this disease especially since α1-tubulin, protein disulfide isomerase and certain tropomyosins have also been implicated in the regulation of neuronal differentiation.

The combination of laser microdissection and two-dimensional gel electrophoresis (2-D PAGE) has been developed to perform proteomic analysis on specific populations of cells in cancer tissues. However, as conventional low sensitivity silver staining was used for spot detection, the microdissection required to obtain an adequate amount of protein for 2-D PAGE is laborious and only a restricted number of protein spots could be visualized. As a consequence, this technology was impractical for direct clinical applications and had a limited impact on cancer studies. To solve these problems, we developed an application in which fluorescent dyes label the proteins extracted from microdissected tissues prior to 2-D PAGE separation. In this application, a small amount of protein, less than 6.6 microg, was enough to generate a 2-D profile with approximately 1500 protein spots. This technique was applied to compare the proteome of normal intestinal epithelium with that of adenoma in Min mice. Thirty-seven protein spots reproducibly showed significant differences in intensities. Mass spectrometric analysis and Western blotting identified eight of them, including prohibitin, 14-3-3zeta, tropomyosin 3 and Hsp84. These results indicate that fluorescence labeling of proteins from microdissected tissues prior to 2-D PAGE is a powerful cancer proteomic study tool.

Anaplastic large-cell lymphoma (ALCL) is a subtype of aggressive non-Hodgkin’s lymphoma first described in 1985, 1 and characterized by the expression of CD30/Ki-1 antigen. By current definition, ALCLs exhibit a T-cell or null phenotype and the majority of cases demonstrate expression of the anaplastic lymphoma kinase (ALK) protein. 2 Most ALK-expressing ALCLs harbor the t(2;5)(p23;q35) chromosomal aberration 3, 4, 5 that involves the 5′-oligomerization motif region of nucleophosmin (NPM) gene on chromosome 5, and the 3′-cytoplasmic tyrosine kinase catalytic domain of anaplastic lymphoma kinase (ALK) gene of chromosome 2 to form the NPM-ALK fusion gene. 6 This rearrangement places the ALK gene under the control of the NPM promoter and results in deregulated expression of the ALK protein. Normal expression of ALK appears to be stringently controlled and limited to the cytoplasm of the testis, ganglion cells of the intestine, and neural tissues. 6 The NPM-ALK fusion protein has been shown by immunohistochemistry to localize in the cytoplasm and the nucleus of the neoplastic cells, thereby providing a distinctive marker for t(2;5)-positive ALCL. The t(2;5)-positive ALCLs account for 80 to 85% of the ALK-positive lymphomas. 7 The remaining 15 to 20% of ALK-positive ALCLs harbor variant fusion partners and exhibit immunohistological patterns different from that observed for t(2;5). 8 In these cases, ALK expression is predominantly cytoplasmic and is not present in a nuclear or nucleolar localization. 8 This is because of ALK forming fusion partners with genes other than NPM. Interestingly, a number of non-t(2;5) translocations have also been identified in a nonhematopoietic soft-tissue neoplasm known as inflammatory myofibroblastic tumor (IMT). 9, 10, 11 Remarkably, IMT may bear a histological resemblance to morphological variants of ALCL such as the lymphohistiocytic, small cell, and sarcomatoid variants. 8, 12 The propensity of ALK for partnering with a diverse variety of genes in both ALCLs and IMTs raises the possibility that other yet unidentified genes that partner with ALK may be involved in the pathogenesis of these tumors. In a quest to identify the ALK fusion partner in a non-t(2,5) ALCL with cytoplasmic expression of the ALK protein, we used the 5′-rapid amplification of cDNA ends (RACE) technique that revealed a fusion between tropomyosin 3 (TPM3) and the ALK genes. Molecular analysis for T-cell clonality also complemented immunohistochemical studies in the definitive assignment of T-cell lineage to the tumor.

Tropomyosin (TM), a component of the thin filament of the sarcomere, is encoded by a four-member multigene family: alpha-TM, beta-TM, TPM 3, and TPM 4. The alpha-TM, beta-TM, and TPM 3 genes each utilize an alternative splicing mechanism to encode a striated muscle isoform. Although the alpha-TM and beta-TM striated muscle isoforms are well characterized, relatively little is known about the TPM 3 isoform. We cloned and sequenced the murine TPM 3 cDNA and found that it exhibits a 93% nucleotide homology and 99% amino acid homology to the human sequence. Results show that, unlike humans, TPM 3 is not expressed in any developmental stage of murine hearts. TPM 3 message is expressed in slow-twitch skeletal muscle but is not found in representative fast-twitch musculature. The soleus, a representative slow-twitch muscle, expresses transcript levels of 65% beta-TM, 15% alpha-TM, and 20% TPM 3, but the TPM 3 protein accounts for approximately 31% of the total striated tropomyosin in slow-twitch muscle. In fast-twitch muscle, alpha-TM comprises 71% of the total striated muscle TM protein, and beta-TM comprises 29%. The results demonstrate that a translational mechanism regulates the production of the TM proteins, with beta-TM message not being efficiently translated. The unique distribution pattern of TPM 3 adds to the diversity of the tropomyosin family and strongly suggests functional significance for the different striated muscle TM isoforms.

The alpha-striated tropomyosin 3' untranslated region (TM UTR) promotes differentiation of fibroblasts into cells resembling skeletal muscle. To investigate the mechanism of this observation, RNA harvested from transfected primary fibroblasts was used for semiquantitative RT-PCR with primers specific for muscle transcription factors, showing that myoD and myogenin transcripts are detected in these cells, but that differentiation after TM UTR expression is independent of a detectable increase in these transcripts. Double immunofluorescent staining with antibodies to myoD family members and to titin confirms that muscle differentiation in TM UTR-transfected fibroblasts is independent of production of any transcription factor in this family. In contrast, the muscle transcription factor myocyte enhancer factor 2 (mef-2) is strongly expressed after transfection of fibroblasts with the TM UTR. The increase in mef-2 protein is due to an increase in the steady-state level of its mRNA, as shown by Northern analysis. The expression of p21 ordinarily observed in skeletal myogenesis before the expression of muscle-specific proteins is not seen in fibroblasts induced to differentiate by the TM UTR. These results demonstrate that post-transcriptional regulation of myoD family members is seen in fibroblasts, and that the TM UTR induces muscle differentiation independent of the myoD transcription factors and without expressing proteins characteristic of terminal withdrawal from the cell cycle. Finally, an increase in the steady-state level of mef-2 transcripts appears in the proximal pathway of myogenic activation in response to expression of the TM UTR. These results imply that fibroblasts can utilize an additional differentiation route upon TM UTR expression resulting in mature muscle other than that requiring myoD family members.

Transfection with a plasmid encoding the 3' untranslated region (3' UTR) of skeletal muscle tropomyosin induces chicken embryonic fibroblasts to express skeletal tropomyosin. Such cells become spindle shaped, fuse, and express titin, a marker of striated muscle differentiation. Skeletal muscle tropomyosin and titin organize in sarcomeric arrays. When the tropomyosin 3' UTR is expressed in osteoblasts, less skeletal muscle tropomyosin is expressed, and titin expression is delayed. Some transfected osteoblasts become spindle shaped but do not fuse nor organize these proteins into sarcomeres. Transfected cells expressing muscle tropomyosin organize muscle and nonmuscle isoforms into the same structures. Thus, the skeletal muscle tropomyosin 3' UTR induces transdifferentiation into a striated muscle phenotype in a cell-type-specific context.

The human tropomyosin 3 (TPM3) gene was previously localized to chromosome 1. The non-muscle isoform of the TPM3 gene product becomes fused to a gene product from the tyrosine kinase receptor gene (NTRKl), previously localized to 1q23→q24, to generate an active oncogene. Two sequence tagged sites spanning three exons and two introns in the carboxy coding region of the gene were used to localize TPM3 to 1q22→q23 by fluorescence in situ hybridization. This localization now places the NTRKl and TPM3 genes in close proximity, so that a gene fusion rearrangement would not be cytologically detected. The 1q22→q23 localization of TPM3 is within the NEM1 locus associated with autosomal dominant nemaline myopathy, making TPM3 a candidate for this disorder.

We have characterized maternal and zygotic cytoskeletal tropomyosin mRNA expression during Drosophila embryogenesis using in situ hybridization to endogenous cytoskeletal tropomyosin mRNA and a cytoskeletal tropomyosin promoter/beta-galactosidase-encoding fusion gene mRNA in transgenic flies. A 2.0-kb maternal cytoskeletal tropomyosin mRNA is synthesized in the nurse cells and transported into the oocyte during oogenesis. During early embryogenesis, this mRNA becomes localized to the pole cell region and then to the cortex during the cellular blastoderm stage. In later embryos it is localized to the ventral and cephalic furrows and extending germ band. The major zygotic mRNA is 2.4 kb and is first detected at gastrulation. In early embryos, this mRNA is expressed in the invaginating anterior and posterior midgut, the transverse furrows, and the amnioserosa, all regions of the embryo undergoing intense cellular movement, and in later embryos, predominantly in the gut, brain, and epidermis. A transgene construct containing 1.2 kb of 5' cytoskeletal tropomyosin promoter sequences driving expression of Escherichia coli beta-galactosidase and the cytoskeletal tropomyosin 3' untranslated region in transgenic flies has the same distribution of maternal and zygotic transcripts throughout oogenesis and embryonic development as the endogenous transcripts. A transgene containing the 3' untranslated region from either the hsp70 gene or the SV40 early genes, on the other hand, does not express maternal RNA. Furthermore, none of the transgenes expressed in the follicle cells, suggesting that expression in these cells is under different transcriptional control. Our results indicate that maternal and zygotic cytoskeletal tropomyosin mRNAs are localized to specific regions of the developing embryo, particularly in regions of the embryo undergoing cell movement and invagination. Furthermore, the synthesis, transport, and accumulation of this RNA is under transcriptional and post-transcriptional control.

Muscle-Specific Transcription Factors in Fibroblasts Expressing the Alpha Striated Tropomyosin 3' Untranslated Region • 124

Seven polypeptides (a, b, c, 1, 2, 3a, and 3b) have been previously identified as tropomyosin isoforms in chicken embryo fibroblasts (CEF) (Lin, J. J.-C., Matsumura, F., and Yamashiro-Matsumura, S., 1984, J. Cell. Biol., 98:116-127). Spots a and c had identical mobility on two-dimensional gels with the slow-migrating and fast-migrating components, respectively, of chicken gizzard tropomyosin. However, the remaining isoforms of CEF tropomyosin were distinct from chicken skeletal and cardiac tropomyosins on two-dimensional gels. The mixture of CEF tropomyosin has been isolated by the combination of Triton/glycerol extraction of monolayer cells, heat treatment, and ammonium sulfate fractionation. The yield of tropomyosin was estimated to be 1.4% of total CEF proteins. The identical set of tropomyosin isoforms could be found in the antitropomyosin immunoprecipitates after the cell-free translation products of total poly(A)+ RNAs isolated from CEF cells. This suggested that at least seven mRNAs coding for these tropomyosin isoforms existed in the cell. Purified tropomyosins (particularly 1, 2, and 3) showed different actin-binding abilities in the presence of 100 mM KCl and no divalent cation. Under this condition, the binding of tropomyosin 3 (3a + 3b) to actin filaments was significantly weaker than that of tropomyosin 1 or 2. CEF tropomyosin 1, and probably 3, could be cross-linked to form homodimers by treatment with 5,5'-dithiobis-(2-nitrobenzoate), whereas tropomyosin a and c formed a heterodimer. These dimer species may reflect the in vivo assembly of tropomyosin isoforms, since dimer formation occurred not only with purified tropomyosin but also with microfilament-associated tropomyosin. The expression of these tropomyosin isoforms in Rous sarcoma virus-transformed CEF cells has also been investigated. In agreement with the previous report by Hendricks and Weintraub (Proc. Natl. Acad. Sci. USA., 78:5633-5637), we found that major tropomyosin 1 was greatly reduced in transformed cells. We have also found that the relative amounts of tropomyosin 3a and 3b were increased in both the total cell lysate and the microfilament fraction of transformed cells. Because of the different actin-binding properties observed for CEF tropomyosins, changes in the expression of these isoforms may, in part, be responsible for the reduction of actin cables and the alteration of cell shape found in transformed cells.