L-plastin Expression Research Articles

Expression and Clinical Significance of l-Plastin in Colorectal Carcinoma

Introductionl-plastin, an actin-binding protein, is upregulated in many tumours, including colorectal carcinoma. This study evaluated the expression of l-plastin in plasma and colorectal tumour tissue and analysed the correlation between clinicopathological staging and prognosis. Materials and MethodsEnzyme-linked immunosorbent assay was used to detect l-plastin in the plasma of 120 colorectal carcinoma patients and 40 control subjects. Immunohistochemistry analyses were also used. ResultsThe rate of positive l-plastin expression was significantly higher in colorectal carcinoma patients than in control subjects, and was significantly higher in tumour tissues than in the tissues surrounding the tumour. l-Plastin expression also is correlated with tumour grade and size, and lymph node metastasis. However, there was no correlation with the extent of tumour invasion or distant metastasis. Conclusionl-Plastin may be a useful marker for screening colorectal carcinoma and determining the prognosis of patients with colorectal carcinoma, and for genetic therapy and targeted therapy of colorectal carcinoma.

A role for the transcription intermediary factor 2 in zebrafish myelopoiesis

TIF2 is fused with MOZ in the inv(8)(p11q13) acute myeloid leukemia. TIF2, member of the p160 family, is a histone acetyl transferase (HAT). Deletion of p160 genes were performed in mice. Some observations suggest that p160 family members may perform overlapping functions in mice. Therefore, we decided to choose the zebrafish model to study TIF2. The aim of this study was to characterize the role of this HAT during embryonic development. We use antisense, morpholino-modified oligomers to transiently knockdown tif2 gene, thus determining whether TIF2 plays a role in zebrafish early development. We show that tif2 is involved in embryogenesis and in primitive hematopoiesis. tif2-knockdown zebrafish embryos are smaller than controls, they demonstrate shorter tails, they display notochord deformation and they exhibit U-shaped tail somites. A synthetic RNA encoding human TIF2 rescues the tif2-knockdown phenotype. Analysis of fli1 expression by whole-mount in situ hybridization indicates normal angioblast specification, but altered localization of intersomitic vessels. The posterior intermediate cell mass, in which a part of primitive hematopoiesis occurs, is altered in tif2 morphants and whole-mount in situ hybridization analyses of l-plastin and mpx expression suggest a specific inhibition of granulocytic and macrophagic differentiation at late stages. These data indicate an important role for TIF2 in zebrafish primitive myelopoiesis.

Analysis of the expression of biomarkers in urinary bladder cancer using a tissue microarray

Dysregulation of Akt, PTEN, Drg-1, Cx-26, and L-plastin expression appear to be important in the progression of various cancers. Their expression in bladder cancer has not been well characterized. To assess the expression of these genes and their relationship to the outcome of bladder cancer, we used a bladder cancer tissue microarray (TMA) of 251 transitional cell carcinomas. We quantitated immunohistochemical staining of each protein using both automated and manual methods and correlated the expression levels with the clinicopathologic characteristics of the tumor and patient survival. Overall, the results from both automated and manual analyses were similar. We found a significant correlation between the expression of PTEN, Cx-26 and L-plastin with known clinically important pathologic features of bladder cancer (tumor grade, stage, and growth pattern). Aberrant localization patterns of Cx-26 and Drg-1 were observed in bladder tumors. There was also a significant correlation in expression among pAkt, PTEN, and L-plastin. Although the expression of these genes correlated with factors known to be associated with patient outcome, none of them was an independent predictor of progression-free or overall survival.

Phosphorylation of ectopically expressed L‐plastin enhances invasiveness of human melanoma cells

The leukocyte specific actin-binding protein L-plastin is aberrantly expressed in several nonhematopoetic malignant tumors. However, little is known about the functional consequences of L-plastin expression. Here, we investigated the function of L-plastin in human malignant melanoma cells. Knock-down of endogenous L-plastin by siRNA treatment reduced migration of the melanoma cell line IF6. However, in melanoma patients, no correlation existed between L-plastin expression and tumor stages. This implied that additional factors such as phosphorylation of L-plastin may influence its function in tumor cells. To investigate this further, EGFP-tagged wild-type L-plastin (wt-LPL-EGFP) and a mutated, nonphosphorylatable L-plastin protein (5A7A-LPL-EGFP), were expressed in the L-plastin negative melanoma cell line MV3. Biochemical analysis revealed that wt-LPL-EGFP is phosphorylated in MV3 cells while 5A7A-LPL-EGFP is not. Although both wt-LPL-EGFP and 5A7A-LPL-EGFP were targeted to, and promote the formation of, vinculin-containing adhesion sites, static adhesion to either Matrigel or isolated extracellular matrix molecules was neither influenced by expression of wt-LPL-EGFP nor by expression of 5A7A-LPL-EGFP when compared with EGFP expressing control cells. In contrast, haptotactic, but not chemotactic, migration of melanoma cells towards either Matrigel or isolated extracellular matrix molecules was similarly enhanced, if either 5A7A-LPL-EGFP or wt-LPL-EGFP were expressed in MV3 cells. Interestingly, only cells expressing the phosphorylatable wt-LPL-EGFP protein showed enhanced invasion into Matrigel. In line with these findings the in vivo metastatic capacity of mouse B16 melanoma cells correlates with expression and phosphorylation of L-plastin. These data show that an increase in melanoma cell invasiveness requires not only expression but also phosphorylation of L-plastin.

A Role for fbxo5 in Zebrafish Myelopoiesis.

The zebrafish is a powerful model system for investigating embryonic vertebrate hematopoiesis that allows for the critical in vivo analysis of cell development and lineage determination. Conservation of the variety of blood cell types and key regulatory factors between mammals and zebrafish suggests that the regulatory mechanisms directing lineage specification are similarly conserved. To identify novel genes necessary for normal myeloid cell development, we have analyzed a panel of zebrafish insertional mutants, provided by Nancy Hopkins at Massachusetts Institute of Technology (MIT), and identified mutants with deficient or abnormal distribution of myeloperoxidase (mpo), a gene specifically expressed in zebrafish granulocytes. One of the mutants identified in this screen is disrupted in fbxo5, a gene not previously identified as having a role in hematopoiesis. Using whole mount RNA in situ analysis on mutant and morpholino-injected embryos (morphants) we have determined that decreased levels of fbxo5 result in severely diminished numbers of granulocytes, although erythropoiesis appears normal. Interestingly, fbxo5 morphants have normal levels of pu.1 and l-plastin expression. Since we have previously shown that knockdown of pu.1 results in the loss of mpo-expressing cells, our data suggest that fbxo5 may function downstream or in a parallel pathway to pu.1 during granulopoiesis. To determine if the decreased number of granulocytes was due to programmed cell death, we examined the level of apoptosis in morphant versus control embryos. Whole mount acridine orange staining of apoptotic cells indicated that fbxo5 morphants had increased apoptosis, which can be mostly rescued by co-injection with p53 morpholino. However, the fbxo5/p53 double morphants continued to display a severe decrease in granulocytes, suggesting that the loss of granulocytes is not due to p53-mediated apoptosis. Fbxo5 has been examined previously in Xenopus oocytes, where the protein was found to regulate cell cycle progression by inhibiting APC. Interestingly, we have identified multiple mutants in genes that normally regulate the G2/M checkpoint and mitosis during the cell cycle all which display decreased numbers of granulocytes, suggesting a role for the regulation of the cell cycle in granulopoiesis. While further analysis is needed to determine where and how fbxo5 functions during myeloid cell development, as well as each of the genes disrupted in our additional cell cycle mutants, we have identified that fbxo5 is necessary specifically for the normal development of granulocytes.

Mutation in the Zebrafish Homolog of the RNA Helicase DHX8 Result in Hematopoietic Defects.

Hematopoiesis is a tightly regulated process that requires the coordinated efforts of many tissue-specific transcription factors. Lateral mesoderm tissue undergoes a series of restricted proliferation and differentiation that result in the production of the hematopoietic stem cells. These cells then differentiate to populate the different cell lineages. While some factors essential for this process have been defined, many remain unknown. Zebrafish offer a powerful model system in which it is possible to conduct forward phenotype-driven screening. We have undertaken a chemical mutagenesis screen using N-nitroso-N-ethylurea (ENU) in the zebrafish to uncover mutants in myelopoiesis and early hematopoiesis. Adult male fish were exposed to ENU in a protocol designed to mutagenize spermatagonia with a mutation frequency of approximately 1:1000. Mutagenized males were crossed to wild-type females to generate F1 females. Haploid embryos from F1 females were screened at 27 hours of development by RNA in situ hybridization using the myeloid-specific marker, leukocyte-specific plastin (l-plastin), and the stem cell marker, core binding factor b (cbfb). One mutant line identified from this screen, mummy, showed reduced cbfb expression and lack of l-plastin expression. Additional in situ staining has confirmed that mummy has a myeloid defect as other myeloid markers such as Lys.C, Mpo, and C/ebp1 were also significantly reduced. Mummy mutant also have a degreased expression of gata1 and bE1 globin (RBC markers) as well as the stem cell marker scl. These results suggested that the defect in mummy is at or up-stream of the HSC level. The mummy mutant embryos also suffer from widespread cell death and die around 36 hours post fertilization. Genetic mapping and positional cloning has identified the mutated gene as the zebrafish homolog of DHX8, which encodes an RNA helicase. The yeast homolog of dhx8, prp22, functions during mRNA splicing and RNA processing. Morpholino injection into wild-type embryos has confirmed that reduced expression of dhx8 can produce a similar phenotype. The wild-type zebrafish dhx8 cDNA but not the mutated dhx8 cDNA identified in mummy was able to partially rescue the mummy phenotype. Preliminary data from RT-PCR and microarray analysis suggest that mummy mutants have defects in the production of spliced transcripts of some genes but not others. Genes with decreased spliced transcripts in the mummy mutant include scl, cbfb, and gata1, which may explain the hematpoietic defects observed. Our results demonstrate that mutations in an RNA helicase involved in splicing could result in specific blockage of hematopoiesis.

Adenoviral vectors with E1A regulated by tumor-specific promoters are selectively cytolytic for breast cancer and melanoma.

We have previously demonstrated that a truncated form of the L-plastin promoter can confer tumor-specific patterns of expression on replication-incompetent adenoviral vector reporter and therapeutic transcription units. In this report, a 2.5-kb truncated version of the L-plastin promoter was placed 5' to the E1A gene of a wild-type adenovirus. The vector generated (Ad-Lp-E1A) was directly cytotoxic to established breast and ovarian cancer cell lines and to primary explant cultures derived from ovarian cancer, but was not cytotoxic to explant cultures of normal mammary epithelial cells. This vector was not cytotoxic to cell lines in which the L-plastin E1A transcription unit was not expressed, whereas the same cell lines were sensitive to the cytotoxic effect of a replication-competent adenoviral vector in which the cytomegalovirus (CMV) promoter drove E1A expression. When the tyrosinase promoter/enhancer was placed 5' to the E1A gene in the adenoviral backbone, the resulting vector (Ad-Tyr-E1A) was selectively toxic to melanoma cells and one percent as toxic to explants of ovarian cancer cells as the Ad-Lp-E1A vector. Injection of these vectors (Ad-Lp-E1A and Ad-Tyr-E1A) into nodules derived from the MCF-7 and MDA-MB-468 human breast cancer cell lines and the TF-2 human melanoma cell line, respectively, which were growing subcutaneously in severe combined immunodeficiency (SCID) mice, induced regression of these tumors. Such vectors may therefore be useful in cancer treatment.

Identification of I-plastin, a human fimbrin isoform expressed in intestine and kidney.

The complete cDNA sequence of human intestine-specific plastin (I-plastin) was determined from a clone derived by PCR. It consists of a 97-bp 5' untranslated region, a 1,887-bp coding region, and a 1,655-bp 3' untranslated region. The coding region predicts a 629-residue polypeptide whose sequence displays 86, 75, and 73% identities with chicken intestine fimbrin, human T-plastin, and human L-plastin, respectively. Recombinant I-plastin cross-linked actin filaments into bundles in the absence but not in the presence of calcium. The I-plastin gene was mapped by PCR to human chromosome 3; the L- and T-plastin genes were previously mapped to chromosomes 13 and X, respectively. I-plastin mRNA was detected in the small intestine, colon, and kidneys; relatively lower levels of expression were detected in the lungs and stomach. In contrast, L-plastin expression was restricted to the spleen and other lymph node-containing organs, while T-plastin was expressed in a variety of organs, including muscle, brain, uterus, and esophagus. In contrast to the situation for the intestine, high levels of L- and T-plastin mRNAs were detected in Caco-2, a human colon-derived cell line. Immunofluorescence microscopy detected I-plastin in the brush border of the small intestine and colon. These results identify I-plastin as the human homolog of chicken intestine fimbrin and as a third plastin isoform in humans.

Human plastin genes. Comparative gene structure, chromosome location, and differential expression in normal and neoplastic cells.

Plastins are a family of actin-binding proteins that are conserved throughout eukaryote evolution and expressed in most tissues of higher eukaryotes. In humans, two ubiquitous plastin isoforms (L and T) have been identified. The L isoform is expressed only in hemopoietic cell lineages, while the T isoform has been found in all other normal cells of solid tissues that have replicative potential (fibroblasts, endothelial cells, epithelial cells, melanocytes, etc.). However, L-plastin has been found in many types of malignant human cells of non-hemopoietic origin suggesting that its expression is induced accompanying tumorigenesis in solid tissues. To learn more about the nature of plastin genes and their potential role in malignancy, the L- and T- plastin genes were cloned and sequenced to characterize their structure and mechanisms of regulation of expression. Each gene was found to be approximately 90 kilobases in size and was composed of 16 exons. All exon-intron junction sequences were identified and shown to conform to the canonical junction sequences. It was evident from their similar structure and coding homology that the two plastin genes have diverged from a common ancestor gene. L- and T-plastin genes were also mapped to chromosomes 13 and X, respectively, using polymerase chain reaction amplification with isoform-specific probes. An expanded survey of normal cell types and 50 tumor cell lines, demonstrated that 68% of carcinomas and 53% of other solid tumors of nonepithelial origin exhibited L-plastin expression, whereas the normal stem cell progenitors did not. Fibrosarcomas (n = 4), ovarian carcinomas (n = 9), breast carcinomas (n = 4), and choriocarcinomas (n = 2) combined exhibited the highest frequency and levels of L-plastin expression (95% frequency). In addition, 4 tumor cell lines that were L-plastin-negative exhibited evidence of defective T-plastin expression increasing the apparent co-incidence of plastin abnormalities associated with human tumorigenesis to 71%. Evidence is presented in support of a trans-activation mechanism for activation of L-plastin synthesis accompanying tumorigenesis. The induction of L-plastin expression accompanying SV40-mediated transformation of human embryonic lung MRC-5 fibroblasts was also confirmed. Finally, we present evidence that fimbrin is a third distinct plastin isoform which is specifically expressed at high levels in the small intestine.

Characterization of the human L-plastin gene promoter in normal and neoplastic cells.

Plastins are a family of human actin-binding proteins (isoforms) which are abundantly expressed in all normal replicating mammalian cells. One isoform, L-plastin, is constitutively expressed at high levels in hemopoietic cell types while T-plastin is constitutively expressed in all non-hemopoietic cells of solid tissues that have replicative potential (fibroblasts, endothelial cells, epithelial cells, melanocytes, etc.). L-plastin is, however, constitutively synthesized in many types of malignant human cells of solid tissues suggesting that its expression is induced during tumorigenesis. The frequency of L-plastin induction in some cancers of the steroid-regulated female reproductive tract (breast, ovary, uterus, and placenta) appears to be especially high (79% in a limited survey). To learn the mechanism of L-plastin gene activation accompanying tumorigenesis, we have begun to characterize the promoter and regulatory elements of the L-plastin gene. Transcription initiation from this promoter was found to occur at multiple sites and as near as 10 base pairs from the 3'-side of the TATAAA box. The promoter and its flanking DNA were cloned and sequenced to identify potential regulatory elements that participate in the induction of the L-plastin gene in neoplastic cells. Examination of upstream sequences revealed the existence of two potential progesterone, one potential estrogen, and four potential Ets-1 responsive elements flanking the promoter. A 315-base pair fragment spanning the TATAAA box and a potential Sp1-binding site exhibited maximum promoter activity using CAT as a reporter while longer promoter fragments extending into upstream flanking sequences spanning the hormone receptor-response elements exhibited reduced promoter activity. An expression vector, pHLPPr-1-neo, was constructed using a 5.1-kilobase pair EcoRI-HindIII fragment of the L-plastin gene that contained the potential upstream regulatory elements, the TATAAA box, and part of the first exon. This promoter could direct the constitutive expression of the reporter beta-galactosidase at high frequency in transfected colonies of transformed cells that express L-plastin constitutively; by contrast, this promoter was virtually inactive in transfected colonies of normal fibroblasts and it exhibited a low frequency of constitutive activation in transfected colonies of in vitro SV40-transformed fibroblasts which did not exhibit L-plastin expression. The utility of this recombinant promoter in determining the mechanism(s) that leads to activation of the L-plastin gene in tumor cells is discussed. The potential significance of regulation of the L-plastin gene by reproductive hormones in cancers arising in hormone-responsive tissues is also discussed.