LRP::FLAG Reduces Phosphorylated Tau Levels in Alzheimer's Disease Cell Culture Models.

BACKGROUND: Alzheimer's disease (AD) is characterized by amyloid-ß (Aß) plaque and neurofibrillary tangle formation, respectively. Neurofibrillary tangles form as a result of the intracellular accumulation of hyperphosphorylated tau. Telomerase activity and levels of the human reverse transcriptase (hTERT) subunit of telomerase are significantly decreased in AD. Recently, it has been demonstrated that the 37 kDa/67 kDa laminin receptor (LRP/LR) interacts with telomerase and is implicated in Aß pathology. Since both LRP/LR and telomerase are known to play a role in the Aß facet of AD, we hypothesized that they might also play a role in tauopathy. OBJECTIVE: This study aimed to determine if LRP/LR has a relationship with tau and whether overexpression of LRP::FLAG has an effect on tauopathy-related proteins. METHODS: We employed confocal microscopy and FRET to determine whether LRP/LR and tau co-localize and interact. LRP::FLAG overexpression in HEK-293 and SH-SY5Y cells as well as analysis of tauopathy-related proteins was assessed by western blotting. RESULTS: We demonstrate that LRP/LR co-localizes with tau in the perinuclear cell compartment and confirmed a direct interaction between LRP/LR and tau in HEK-293 cells. Overexpression of LRP::FLAG in HEK-293 and SH-SY5Y cells decreased total and phosphorylated tau levels with a concomitant decrease in PrPc levels, a tauopathy-related protein. LRP::FLAG overexpression also resulted in increased hTERT levels. CONCLUSION: This data suggest that LRP/LR extends its role in AD through a direct interaction with tau, and recommend LRP::FLAG as a possible alternative AD therapeutic via decreasing phosphorylated tau levels.

Incomplete penetrance for isolated congenital asplenia in humans with mutations in translated and untranslated RPSA exons.

Isolated congenital asplenia (ICA) is the only known human developmental defect exclusively affecting a lymphoid organ. In 2013, we showed that private deleterious mutations in the protein-coding region of RPSA, encoding ribosomal protein SA, caused ICA by haploinsufficiency with complete penetrance. We reported seven heterozygous protein-coding mutations in 8 of the 23 kindreds studied, including 6 of the 8 multiplex kindreds. We have since enrolled 33 new kindreds, 5 of which are multiplex. We describe here 11 new heterozygous ICA-causing RPSA protein-coding mutations, and the first two mutations in the 5'-UTR of this gene, which disrupt mRNA splicing. Overall, 40 of the 73 ICA patients (55%) and 23 of the 56 kindreds (41%) carry mutations located in translated or untranslated exons of RPSA. Eleven of the 43 kindreds affected by sporadic disease (26%) carry RPSA mutations, whereas 12 of the 13 multiplex kindreds (92%) carry RPSA mutations. We also report that 6 of 18 (33%) protein-coding mutations and the two (100%) 5'-UTR mutations display incomplete penetrance. Three mutations were identified in two independent kindreds, due to a hotspot or a founder effect. Finally, RPSA ICA-causing mutations were demonstrated to be de novo in 7 of the 23 probands. Mutations in RPSA exons can affect the translated or untranslated regions and can underlie ICA with complete or incomplete penetrance.

INS-1 cell glucose-stimulated insulin secretion is reduced by the downregulation of the 67 kDa laminin receptor.

Understanding ß cell-extracellular matrix (ECM) interactions can advance our knowledge of the mechanisms that control glucose homeostasis and improve culture methods used in islet transplantation for the treatment of diabetes. Laminin is the main constituent of the basement membrane and is involved in pancreatic ß cell survival and function, even enhancing glucose-stimulated insulin secretion. Most of the studies on cell responses towards laminin have focused on integrin-mediated interactions, while much less attention has been paid on non-integrin receptors, such as the 67 kDa laminin receptor (67LR). The specificity of the receptor-ligand interaction through the adhesion of INS-1 cells (a rat insulinoma cell line) to CDPGYIGSR-, GRGDSPC- or CDPGYIGSR + GRGDSPC-covered surfaces was evaluated. Also, the effects of the 67LR knocking down over glucose-stimulated insulin secretion were investigated. Culture of the INS-1 cells on the bioactive surfaces was improved compared to the low-fouling carboxymethyl dextran (CMD) surfaces, while downregulation of the 67LR resulted in reduced cell adhesion to surfaces bearing the CDPGYIGSR peptide. Glucose-stimulated insulin secretion was hindered by downregulation of the 67LR, regardless of the biological motif available on the biomimetic surfaces on which the cells were cultured. This finding illustrates the importance of the 67LR in glucose-stimulated insulin secretion and points to a possible role of the 67LR in the mechanisms of insulin secretion.

37-kDa laminin receptor precursor promotes lung adenocarcinoma cell invasion and metastasis by epithelial-to-mesenchymal transition.

37-kDa laminin receptor precursor (37LRP) has a crucial role in migration of some human cancers. Epithelial-to-mesenchymal transition (EMT) has received much attention in invasion and metastasis of lung cancer. Nevertheless, the role of 37LRP is not entirely clear in EMT promotion of lung cancer at present. In this study, we firstly examined the possible role of 37LRP in the invasiveness and metastasis process of lung cancer using immunohistochemistry of 80 lung adenocarcinoma cases, western blot and real-time PCR of 12 fresh lung adenocarcinoma tissues. The results showed that 37LRP significantly correlated with clinical stage and were highly expressed in metastatic lung adenocarcinomas compared with nonmetastatic ones. In vitro, we observed that 37LRP significantly increased the adhesive, invasive and metastatic abilities of human lung adenocarcinoma cell lines A549 by 37LRP-lentivirus interference. Furthermore, inoculation of A549 cells transduced with 37LRP-lentivirus in nude mice resulted in multi-metastases including the lung. In addition, western blotting and immunofluorescence were used to detect the significant difference in expression of E-cadherin and fibronectin in A549 by 37LRP-lentivirus interference compared with 37LRP-small interference RNA-lentivirus interference in vitro and vivo. The data indicated that A549 cells of epithelial cell characteristics might be induced to undergo EMT by 37LRP. A549 cells transduced with 37LRP-lentivirus showed marked morphological changes, accompanied by the decrease of epithelial marker E-cadherin and the increase of mesenchymal marker fibronectin. These results indicated that 37LRP may promote lung adenocarcinoma invasion and metastasis via the mechanism of EMT.

Cloning and expression analysis of the 37-kDa laminin receptor precursor gene from Hyriopsis cumingii.

Hyriopsis cumingii is an economically important freshwater pearl mussel with high pearl quality that is endemic in China. Investigation of genes relevant to shell formation is important for increased pearl output. The substances that form mollusk shells are secreted by epithelial cells in the mantle, the proliferation of which influences secretion ability. This study focused on the proliferation-related 37-kDa laminin receptor precursor (37LRP) of H. cumingii. The full-length cDNA (1133 bp) encoding this 300-amino acid protein was cloned from the mantle. Quantitative fluorescence analysis showed that 37LRP expressed in eight tissues, with the highest expression observed in the liver, and its expression pattern in the mantle reflected shell repair. During repair, 37LRP expression was higher in the experimental shell repair group than that in the control group, exhibiting an initial increase followed by a decrease in expression, and returning to basal levels on completion of the repair. A similar trend was also observed with respect to immunity and cellular metabolism. Expression of the 37LRP protein in the experimental group was significantly higher than that in the control group at the first and second days after shell injury. After 4 days, 37LRP expression in the experimental group was lower than that in the control group. In situ hybridization revealed a strong positive signal corresponding to the 37LRP mRNA at the horny grooves of the mantle, evagination, and in epithelial cells of the velum, which implicated these areas in the repair and formation of the cuticle, prismatic layer, and nacre.

Expression of 67-kDa laminin receptor was associated with tumor progression and poor prognosis in epithelial ovarian cancer.

OBJECTIVE: 67-kDa laminin receptor (67LR) has been identified as a prognostic biomarker for a variety of human cancers. We investigated the clinical significance of 67LR expression and its functional role in epithelial ovarian cancer (EOC). METHODS: 67LR expression was evaluated by immunohistochemistry in 62 patients with EOC. We assessed the correlation of 67LR expression with clinical characteristics. In vitro experiment was performed for 67LR with inhibition using siRNA to evaluate its role in cell survival, apoptosis, and invasion in EOC cells. RESULTS: 67LR was predominantly expressed on the cell membrane in the majority of EOC samples (45/62, 73%). 67LR expression was significantly correlated with advanced stage (P=0.001). Patients with 67LR expression had shorter progression-free survival among all the patients (P=0.010) and in particular among patients with advanced stages (P=0.046). When 67LR expression was inhibited by siRNA in EOC cells (HeyA8 and A2780), there was a significant decrease of cell proliferation and invasion as well as increase of apoptosis. CONCLUSION: These findings suggest that 67LR expression may play an important role in tumor progression into advanced stage with poor prognosis in EOC and down-regulation of 67LR on tumor cells may be a therapeutic target in those patients.

The unusual response of serotonergic neurons after CNS Injury: lack of axonal dieback and enhanced sprouting within the inhibitory environment of the glial scar.

Serotonergic neurons possess an enhanced ability to regenerate or sprout after many types of injury. To understand the mechanisms that underlie their unusual properties, we used a combinatorial approach comparing the behavior of serotonergic and cortical axon tips over time in the same injury environment in vivo and to growth-promoting or growth-inhibitory substrates in vitro. After a thermocoagulatory lesion in the rat frontoparietal cortex, callosal axons become dystrophic and die back. Serotonergic axons, however, persist within the lesion edge. At the third week post-injury, 5-HT+ axons sprout robustly. The lesion environment contains both growth-inhibitory chondroitin sulfate proteoglycans (CSPGs) and growth-promoting laminin. Transgenic mouse serotonergic neurons specifically labeled by enhanced yellow fluorescent protein under control of the Pet-1 promoter/enhancer or cortical neurons were cultured on low amounts of laminin with or without relatively high concentrations of the CSPG aggrecan. Serotonergic neurons extended considerably longer neurites than did cortical neurons on low laminin and exhibited a remarkably more active growth cone on low laminin plus aggrecan during time-lapse imaging than did cortical neurons. Chondroitinase ABC treatment of laminin/CSPG substrates resulted in significantly longer serotonergic but not cortical neurite lengths. This increased ability of serotonergic neurons to robustly grow on high amounts of CSPG may be partially due to significantly higher amounts of growth-associated protein-43 and/or ß1 integrin than cortical neurons. Blocking ß1 integrin decreased serotonergic and cortical outgrowth on laminin. Determining the mechanism by which serotonergic fibers persist and sprout after lesion could lead to therapeutic strategies for both stroke and spinal cord injury.

Changes in cell migration-related molecules expressed by thymic microenvironment during experimental Plasmodium berghei infection: consequences on thymocyte development.

We previously showed alterations in the thymus during experimental infection with Plasmodium berghei. Such alterations comprised histological changes, with loss of cortical-medullary limits, and the intrathymic presence of parasites. As the combination of chemokines, adhesion molecules and extracellular matrix (ECM) is critical to appropriate thymocyte development, we analysed the thymic expression of ECM ligands and receptors, as well as chemokines and their respective receptors during the experimental P. berghei infection. Increased expression of ECM components was observed in thymi from infected mice. In contrast, down-regulated surface expression of fibronectin and laminin receptors was observed in thymocytes from these animals. Moreover, in thymi from infected mice there was increased CXCL12 and CXCR4, and a decreased expression of CCL25 and CCR9. An altered thymocyte migration towards ECM elements and chemokines was seen when the thymi from infected mice were analysed. Evaluation of ex vivo migration patterns of CD4/CD8-defined thymocyte subpopulations revealed that double-negative (DN), and CD4(+) and CD8(+) single-positive (SP) cells from P. berghei-infected mice have higher migratory responses compared with controls. Interestingly, increased numbers of DN and SP subpopulations were found in the spleens of infected mice. Overall, we show that the thymic atrophy observed in P. berghei-infected mice is accompanied by thymic microenvironmental changes that comprise altered expression of thymocyte migration-related molecules of the ECM and chemokine protein families, which in turn can alter the thymocyte migration pattern. These thymic disturbances may have consequences for the control of the immune response against this protozoan.

Differentiation-associated alteration in sensitivity to apoptosis induced by (-)-epigallocatechin-3-O-gallate in HL-60 cells.

Green tea and its constituent (-)-epigallocatechin-3-O-gallate (EGCG) are known to have apoptosis-inducing activity on tumor cells including human leukemia HL-60 cells, providing an explanation for their anti-cancer effects. In the present study, we compared the sensitivity of undifferentiated cells and differentiated HL-60 cells with normal-like phenotypic characters. HL-60 cells treated with three differentiating agents were found to be resistant to EGCG-mediated apoptosis as compared with undifferentiated cells. Gene and protein expression of 67 kDa laminin receptor was down-regulated in differentiated HL-60 cells, suggesting its contribution to the difference in sensitivity in view of the fact that the receptor is a target of EGCG's action to induce apoptosis. The finding supports the view that EGCG induces apoptosis preferentially in cancer cells as compared with normal counterparts.

Expression of laminin receptor 1 in gestational trophoblastic diseases and normal placenta and its relationship with the development of postmolar tumors.

OBJECTIVES: To investigate the expression of laminin receptor 1 (LR1), a non-integrin-type laminin receptor, in gestational trophoblastic diseases and normal first-trimester placenta, since it may play a role in controlling trophoblast invasion in normal and molar pregnancies. METHODS: Paraffin sections from 24 gestational age controlled normal first-trimester placentas, 47 partial moles, 56 complete moles, 3 invasive moles, 4 gestational choriocarcinomas, and 1 placental-site trophoblastic tumor were studied immunohistochemically for expression of LR1. RESULTS: In complete and partial moles, decidual cells showed significantly stronger LR1 protein staining compared to the normal placenta (p<0.01). When compared to the partial moles, weak staining in less than 33% of decidual cells was also more prominent in the normal placenta (p<0.05). Complete and partial moles, invasive moles, choriocarcinomas, and placental-site tumors did not differ from each other with respect to staining intensity. Strong immunostaining for LR1 in decidual cells, cytotrophoblasts, syncytiotrophoblasts, and extracellular matrix cells of partial and complete moles was not significantly correlated with the development of persistent postmolar gestational trophoblastic tumors. CONCLUSIONS: LR1 may be important in the pathogenesis of gestational trophoblastic diseases. The increased expression of LR1 in decidual cells of partial and complete moles may not influence the development of persistent gestational trophoblastic tumor. Since they are seen rarely, multicentric studies should be planned to study LR1 expression in invasive moles and gestational trophoblastic neoplasms.

Overexpression of malignancy-associated laminins and laminin receptors by angiotropic human melanoma cells in a chick chorioallantoic membrane model.

BACKGROUND: As distinct from intravascular/lymphatic dissemination, extravascular migratory metastasis (EVMM) has been described as a potential additional mechanism of melanoma spread in which tumor cells migrate along the external surfaces of vessels. Angiotropic melanoma cells are linked to the endothelium by a matrix containing laminin. In addition, it has been shown that C16 laminin-derived peptide increases extravascular migration of human green fluorescent protein (GFP) melanoma cells along vessels in a chicken chorioallantoic membrane model (CAM). In this study, we have tested the hypothesis that expression levels of some genes related to lamimin and metastasis are differentially expressed in vascularized angiotropic melanoma areas vs. avascular melanoma areas from the same tumor. DESIGN: C8161 human melanoma cells in a shell-less chick CAM assay were used to study EVMM associated with the presence of vascularized angiotropic melanoma areas. For both high-quality histomorphology and RNA preservation in paraffin-embedded tissue, we used a methanol-based fixative coupled with microwave-assisted rapid tissue processing as previously described. Using laser capture microdissection, angiotropic melanoma areas as well as avascular areas were microdissected. Using quantitative real time polymerase chain reaction (QRT-PCR), six genes have been studied: LAMC2 (laminin gamma2 chain), LAMA4 (laminin alpha4 chain), ITGB1 (integrin beta1), ITGB3 (integrin beta3), RSPA (ribosomal protein), and MMP2 (matrix metallopeptidase 2). QRT-PCR data were normalized to human GAPDH housekeeping gene and values were compared against Human Total RNA. Final results were expressed as percentage of expression. RESULTS: All tumors demonstrated a similar pattern, i.e. EVMM of angiotropic melanoma cells. The microdissected histopathological sections presented both angiotropic areas and avascular areas. All genes were overexpressed in angiotropic melanoma areas vs. avascular melanoma areas, especially LAMC2, LAMA4 and ITGB3 (respectively, 165.18, 208.86, and 483.69%). CONCLUSION: This study shows that several genes related to laminin are overexpressed in angiotropic melanoma areas vs. avascular melanoma areas. Since extravascular migration of melanoma cells along vessels has been demonstrated in the CAM model, taken together these results suggests that some laminins and laminin receptors may play a role in extravascular migratory metastasis. This model may represent a promising strategy to analyze differential gene expression in EVMM.

Susceptibility of colorectal cancer cells to Sindbis virus infection.

Sindbis virus (SIN), a member of the Togaviridae family, infects a broad range of cells and has been shown to be an effective anti-tumor agent. The infection efficiency of the virus, however, varies greatly among target cells. In this report, we compared the ability of SIN to infect colorectal cancer cells and cells of other cancer origin. While tumor cells from breast, leukemia, and prostate cancers were largely resistant to SIN infection, nine of the ten colorectal cancer cell lines tested were sensitive to SIN infection. Moreover, SIN susceptibility correlated with the metastatic potential of the colorectal cancer cells. Two highly aggressive and invasive cell lines, SW620 and COLO-320DM were the most sensitive to SIN infection. Similarly, SIN preferentially targeted metastatic tumor cells in a mouse xenograft model for colon cancer progression. The higher infection rate was not due to increased expression of the 67kD laminin receptor, a specific receptor for SIN infection, although viral attachment and entry were markedly enhanced in SW620 cells. These results suggest that SIN may employ a novel cell attachment/entry mechanism during infection, allowing selective targeting of colorectal cancer cells.

Overexpression of laminin receptor 1 on decidual cells in partial and complete mole.

OBJECTIVES: Laminin receptor 1 (LR1), a non-integrin-type laminin receptor, has been described in several tumors and may play a role in tumor invasion. It is well known that LR1 can modify the conformation and degradation of laminin thus enhancing tumor cell invasion. LR1 may play a role in controlling trophoblast invasion in normal and molar pregnancies. MATERIALS AND METHODS: Real-time polymerase chain reaction (RT-PCR) was performed on total cDNA from 17 gestational age-controlled normal placentas, 10 complete moles (CM), and 4 partial moles (PM). Immunolocalization of LR1 was performed on paraffin sections prepared from 17 age-controlled placentas, 17 PM, and 19 CM. RESULTS: RT-PCR demonstrated a 13-fold increase in LR1 mRNA expression in molar tissues (PM and CM combined) versus normal placentas (p=0.012). Immunohistochemical analysis revealed LR1 localized to the decidual cells. In normal placenta LR1 localized to the decidual cell membrane. However, in PM and CM, LR1 was additionally noted in the cytoplasm of decidual cells. Interestingly, with immunohistochemistry method, we found that PM demonstrated higher protein expression of LR1 than either normal placenta or CM (p=0.001 and p=0.024, respectively). CONCLUSION: LR1 is expressed in both the decidual cells of normal placenta and mole (CM and PM). Decidual cells in CM and PM express LR1 significantly greater than the decidual cells in placenta. The underlying mechanism of how molar tissue may be associated with enhanced expression of LR1 in the maternal endometrium is unclear.

[Expression and activity analysis of human 67kD laminin receptor in Pichia pastoris].

To carry out the secretive expression of human 67 kD laminin receptor (67LR), recombinant expression plasmid pPIC9K-67LR was constructed by inserting of 67LR cDNA into yeast expression vector pPIC9K. The 67LR protein was expressed in Pichia pastoris after induced by methanol, and about 12.56 mg electrophoresis purity 67LR could be obtained after the purification of 1L culture using affinity chromatograph column. In vitro competitive binding assay showed that target protein has an excellent biological activity. The successful expression of 67LR has placed a solid foundation for the research on structure and functions of 67LR.

Identification of HLA-A*0201-presented T cell epitopes derived from the oncofetal antigen-immature laminin receptor protein in patients with hematological malignancies.

The oncofetal Ag immature laminin receptor (OFA-iLR) is a potential target molecule for immunotherapeutic studies in several tumor entities, including hematological malignancies. In the present study, we characterize two HLA-A*0201-presented epitopes eliciting strong OFA-iLR peptide-specific human cytotoxic T cell (CTLs) responses in vitro. Both allogeneic HLA-A*0201-matched and autologous CTLs recognized and killed endogenously OFA-iLR-expressing tumor cell lines and primary malignant cells from patients with hemopoietic malignancies in an MHC-restricted fashion but spared nonmalignant hemopoietic cells. Spontaneous OFA-iLR peptide-specific T cell reactivity was detectable in a significant proportion of leukemia patients. Interestingly, in patients with chronic lymphocytic leukemia and multiple myeloma but not in those with acute myeloid leukemia, significant frequencies of OFA peptide-specific CTLs could be detected in an early stage of disease but disappeared in patients with progressive disease. The identification of OFA-iLR-derived peptide epitopes provides a basis for tumor immunological studies and therapeutic vaccination strategies in patients with OFA-iLR-expressing malignancies.

The p67 laminin receptor identifies human erythroid progenitor and precursor cells and is functionally important for their bone marrow lodgment.

The laminins are a group of extracellular matrix proteins with constitutive expression in all tissues, including bone marrow stroma. A functional role for the nonintegrin laminin receptor p67 has been described for cancer metastasis and lymphocyte trafficking. Expression of p67 was also reported for other subsets of mature leukocytes and for malignant hematopoietic or nonhematopoietic cells. We explored p67 expression on normal hematopoietic progenitor cells (HPCs) and its putative role in bone marrow retention of transplanted HPCs. We found p67 expression on a subset of primary human CD34(+) cells coexpressing erythroid markers. Of importance, p67 recognizes early erythroid progenitors, since sorted p67(+) cells were significantly enriched for burst-forming units-erythroid (BFU-Es) and depleted of colony-forming units--granulocyte/macrophage (CFU-GMs). Blockade of p67 binding of donor cells, using antifunctional antibody, reduced bone marrow homing of BFU-Es. These studies identify p67 as a novel phenotypic marker for erythroid HPCs of functional importance for lineage-specific homing/retention among adult transplanted HPCs.

Proteins differentially expressed in response to nicotine in five rat brain regions: identification using a 2-DE/MS-based proteomics approach.

To determine protein expression patterns within the central nervous system (CNS) in response to nicotine, 2-DE/MS was performed on samples from five brain regions of rats that had received nicotine bitartrate by osmotic minipump infusion at a dose of 3.15 mg/kg/day for 7 days. After spot matching and statistical analysis, 41 spots in the amygdala, 49 in the nucleus accumbens (NA), 46 in the prefrontal cortex (PFC), 36 in the striatum, and 28 in the ventral tegmental area (VTA) showed significant differences in the nicotine-treated compared with control samples. Using MALDI-TOF MS peptide fingerprinting, 14 proteins in the amygdala, 11 in the NA, 19 in the PFC, 13 in the striatum, and 19 in the VTA were identified. Several proteins (e.g. dynamin 1, laminin receptors, aldolase A, enolase 1 alpha, Hsc70-ps1, and N-ethylmaleimide-sensitive fusion protein) were differentially expressed in multiple brain regions. By gene ontology analysis, these differentially expressed proteins were grouped into biological process categories, such as energy metabolism, synaptic function, and oxidative stress metabolism. These data, in combination with microarray analysis of mRNA transcripts, have the potential to identify the CNS gene products that show coordinated changes in expression at both the RNA and protein levels in response to nicotine.

Characterization of 67 kD laminin receptor, a protein whose gene is overexpressed on treatment of cells with anti-benzo[a]pyrene-7,8-diol-9,10-epoxide.

The molecular mechanisms potentially related to tumorigenesis induced by anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (anti-BPDE) were investigated by suppression subtractive hybridization of the human bronchial epithelial cells (16HBE) carcinoma induced by BPDE-transformed 16HBE cells (16HBE-C). The 67 kD laminin receptor gene (67LR1) is one of the screened overexpressed genes in 16HBE-C cells when compared with 16HBE. In order to understand the main functions of 67LR1 gene, we amplified the full length of 67LR1 gene using reverse transcription-polymerase chain reaction (RT-PCR) method. The amplified gene products were inserted into pcDNA 3.1 Directional TOPO expression vector. We then transfected 16HBE cells with this vector and derived stable transfected 16HBE cell lines containing the 67LR1 gene by using lipofectin and G418 selection protocols. The expression products of transfected genes were analyzed by semiquantitative RT-PCR. Soft agar growth assay was carried out to identify the malignant features of 67LR1 gene. The stable transfected cell lines can form colonies in soft agar. Further, the transfected cells showed morphological changes compared to the control cells, such as the obvious pseudopods. These data suggest that the 67LR1 gene may be related to malignant transformation induced by the anti-BPDE. The 67LR1 protein may be related to the directionality of cell movement.

Genes differentially expressed in responsive and refractory acute leukemia.

DNA microarray in 22 patients with acute leukemia revealed genes which were differentially expressed. Ribosomal protein SA (RPSA), minichromosome maintenance deficient 2 (MCM2) and heterogeneous nuclear ribonucleoprotein A1 (HNRPA1) were significantly upregulated (p<0.05, t test) in refractory patients, suggesting that they may play a role in refractoriness in acute leukemia and could be biomarkers of prognosis.

Cloning and characterization of a novel spermiogenesis-related gene, T6441, in rat testis.

We report in the present study the cloning and characterization of a novel gene, named T6441, initially derived by the suppressive subtracted hybridization (SSH) cDNA library. The full-length T6441cDNA was 664 bp long, containing a complete open-reading frame for a protein of 149 amino acids (aa). The protein bears no homology to any reported genes. It is predicted that the molecular mass was about 16.7 kDa. Northern blot analysis showed that the T6441 gene had about 4 transcripts in adult rat testis and was temporally regulated in a stage-dependent manner in the testis. In situ hybridization showed that T6441 mRNA was specifically localized in spermatids, and its expression level varied in the cells at different stages of the testicular development, with the highest level at steps 7-14. RT-PCR results showed that the T6441 mRNA was transcribed in most of the tested tissues with its strongest signal in the testis. Recombinant T6441 protein was prepared, purified, and was used to raise rabbit. Western blot analysis using the antiserum revealed four possible testicular specific proteins with their molecular weights being about 22, 25, 50 and 55 kDa respectively. The T6441 protein was expressed mainly in the cytoplasm of spermatids with the maximal levels at steps 12-19. At step 19 spermatid, the T6441 was mainly localized in the residual bodies. The cytoplasm localization of T6441 protein was supported by transient over expression of GFP-fusion protein in Hela cells. Interestingly, the expression of T6441 caused death of transfected cells within 48 h. Our preliminary experimental results suggest that the T6441 gene may play a role in cytoplasm movement and removal during spermiogenesis.