BACKGROUND: The calcium-binding protein calretinin (gene name: CALB2) is currently considered as the most sensitive and specific marker for the diagnosis of malignant mesothelioma (MM). MM is a very aggressive tumor strongly linked to asbestos exposure and with no existing cure so far. The mechanisms of calretinin regulation, as well as its distinct function in MM are still poorly understood.
METHODS: We searched for transcription factors binding to the CALB2 promoter and modulating calretinin expression. For this, DNA-binding assays followed by peptide shotgun-mass spectroscopy analyses were used. CALB2 promoter activity was assessed by dual-luciferase reporter assays. Furthermore, we analyzed the effects of CALB2 promoter-binding proteins by lentiviral-mediated overexpression or down-regulation of identified proteins in MM cells. The modulation of expression of such proteins by butyrate was determined by subsequent Western blot analysis. Immunohistochemical analysis of embryonic mouse lung tissue served to verify the simultaneous co-expression of calretinin and proteins interacting with the CALB2 promoter during early development. Finally, direct interactions of calretinin with target proteins were evidenced by co-immunoprecipitation experiments.
RESULTS: Septin 7 was identified as a butyrate-dependent transcription factor binding to a CALB2 promoter region containing butyrate-responsive elements (BRE) resulting in decreased calretinin expression. Accordingly, septin 7 overexpression decreased calretinin expression levels in MM cells. The regulation was found to operate bi-directionally, i.e. calretinin overexpression also decreased septin 7 levels. During murine embryonic development calretinin and septin 7 were found to be co-expressed in embryonic mesenchyme and undifferentiated mesothelial cells. In MM cells, calretinin and septin 7 colocalized during cytokinesis in distinct regions of the cleavage furrow and in the midbody region of mitotic cells. Co-immunoprecipitation experiments revealed this co-localization to be the result of a direct interaction between calretinin and septin 7.
CONCLUSIONS: Our results demonstrate septin 7 not only serving as a "cytoskeletal" protein, but also as a transcription factor repressing calretinin expression. The negative regulation of calretinin by septin 7 and vice versa sheds new light on mechanisms possibly implicated in MM formation and identifies these proteins as transcriptional regulators and putative targets for MM therapy.

Infantile fibrosarcomas (IFS) represent a distinct group of soft tissue tumors occurring in patients under 2 years of age and most commonly involving the extremities. Most IFS show recurrent ETV6-NTRK3 gene fusions, sensitivity to chemotherapy, and an overall favorable clinical outcome. However, outside these well-defined pathologic features, no studies have investigated IFS lacking ETV6-NTRK3 fusions, or tumors with the morphology resembling IFS in older children. This study was triggered by the identification of a novel SEPT7-BRAF fusion in an unclassified retroperitoneal spindle cell sarcoma in a 16-year-old female by targeted RNA sequencing. Fluorescence in situ hybridization screening of 9 additional tumors with similar phenotype and lacking ETV6-NTRK3 identified 4 additional cases with BRAF gene rearrangements in the pelvic cavity (n=2), paraspinal region (n=1), and thigh (n=1) of young children (0 to 3 y old). Histologically, 4 cases including the index case shared a fascicular growth of packed monomorphic spindle cells, with uniform nuclei and fine chromatin, and a dilated branching vasculature; while the remaining case was composed of compact cellular sheets of short spindle to ovoid cells. In addition, a minor small blue round cell component was present in 1 case. Mitotic activity ranged from 1 to 9/10 high power fields. Immunohistochemical stains were nonspecific, with only focal smooth muscle actin staining demonstrated in 3 cases tested. Of the remaining 5 BRAF negative cases, further RNA sequencing identified 1 case with EML4-NTRK3 in an 1-year-old boy with a foot IFS, and a second case with TPM3-NTRK1 fusion in a 7-week-old infant with a retroperitoneal lesion. Our findings of recurrent BRAF gene rearrangements in tumors showing morphologic overlap with IFS expand the genetic spectrum of fusion-positive spindle cell sarcomas, to include unusual presentations, such as older children and adolescents and predilection for axial location, thereby opening new opportunities for kinase-targeted therapeutic intervention.

DNA replicates in a timely manner with each cell division. Multiple proteins and factors are involved in the initiation of DNA replication including a dynamic interaction between Cdc10-dependent transcript (Cdt1) and Geminin (GMNN). A conformational change between GMNN-Cdt1 heterotrimer and heterohexamer complex is responsible for licensing or inhibition of the DNA replication. This molecular switch ensures a faithful DNA replication during each S phase of cell cycle. GMNN inhibits Cdt1-mediated minichromosome maintenance helicases (MCM) loading onto the chromatin-bound origin recognition complex (ORC) which results in the inhibition of pre-replication complex assembly. GMNN modulates DNA replication by direct binding to Cdt1, and thereby alters its stability and activity. GMNN is involved in various stages of development such as pre-implantation, germ layer formation, cell commitment and specification, maintenance of genome integrity at mid blastula transition, epithelial to mesenchymal transition during gastrulation, neural development, organogenesis and axis patterning. GMNN interacts with different proteins resulting in enhanced hematopoietic stem cell activity thereby activating the development-associated genes' transcription. GMNN expression is also associated with cancer pathophysiology and development. In this review we discussed the structure and function of GMNN in detail. Inhibitors of GMNN and their role in DNA replication, repair, cell cycle and apoptosis are reviewed. Further, we also discussed the role of GMNN in virus infected host cells.

Septins are a novel class of GTP-binding cytoskeletal proteins evolutionarily conserved from yeast to mammals and have now been found to play a contributing role in a broad range of tumor types. However, their functional importance in breast cancer remains largely unclear. Here, we demonstrated that pharmaceutical inhibition of global septin dynamics would greatly suppress proliferation, migration and invasiveness in breast cancer cell lines. We then examined the expression and subcellular distribution of the selected septins SEPT2 and SEPT7 in breast cancer cells, revealing a rather variable localization of the two proteins with cell cycle progression. To determine the role of both septins in mediating malignant behavior of cancer cells, we used RNA silencing to specifically deplete endogenous SEPT2 or SEPT7 in highly invasive breast cancer cell line MDA-MB-231. Our findings showed that SEPT2/7 depletion had virtually identical inhibitory effects on cellular proliferation, apoptosis, migration and invasion. Moreover, the opposite performance in migration and invasion was observed after enforced expression of SEPT2/7 in the same cell line. We further demonstrated MEK/ERK activation, but not other MAPKs and AKT, was positively correlated with the protein levels of SEPT2 and SEPT7. Additionally, in SEPT2/7-overexpressing cells, the MEK specific inhibitor U0126 was able to correct the high active status of MEK/ERK while normalizing the increased invasive behaviors of these cells. Taken together, these results strongly suggest that SEPT2 and SEPT7 are involved in breast carcinogenesis and may serve as valuable therapeutic targets for breast cancer.

BACKGROUND: miR-301a is frequently dysregulated and specific to human tumors, playing a critical role in tumorigenesis; however, the exact functions and regulatory mechanisms of miR-301a in glioma cells remain largely unknown. Herein, we show that miR-301a activated by the Wnt/β-catenin pathway promoted the invasion of glioma cells by directly targeting SEPT7.
METHODS: Biochemical, luciferase reporter, and hromatin immunoprecipitation PCR assays characterized the function and regulatory mechanisms of miR-301a in glioma invasion.
RESULTS: Initially, we detected the expression of miR-301a in glioma tissues and identified that miR-301a had increased, with ascending grades of the tumor. Furthermore, high levels of miR-301a were associated with a poorer prognosis in glioma patients. It is important to note that the Wnt/β-catenin/TCF4 pathway enhanced miR-301a expression by binding to the promoter region. To determine the oncogenic functions of miR-301a in glioma, SEPT7 was supported as the direct target gene. In addition, the Wnt/β-catenin pathway repressed SEPT7 expression, which was dependent on miR-301a in glioma cells. Finally, miR-301a was activated by Wnt/β-catenin and then promoted invasion of glioma cells by inhibiting the expression of SEPT7 in vitro and in vivo.
CONCLUSIONS: Our findings revealed the mechanism of action for miR-301a in tumor cell invasion. Moreover, the Wnt/miR-301a/SEPT7 signaling axis might be a novel target in treating glioma.

Glioma cell metastasis is a serious obstacle for surgical treatment and prognosis, of which locomotion of the cytoskeleton is a key contributor of cancer cell spreading. SEPT7 is documented as a cytoskeletal protein with GTPase activity and involved in glioma progression. However, the underlying mechanism of SEPT7 in glioma invasion remains unresolved. Our study investigated whether SEPT7 influences glioma cell migration involved in cytoskeleton modulation. The SEPT7 expression in various glioma cell lines was markedly decreased compared to in normal human brain cells. It was demonstrated that SEPT7 overexpression significantly inhibits LN18 cell migration and chemotaxis induced by IGF‑1 (P<0.01 and P<0.01). Moreover, MMP‑2 and MMP‑9 were dramatically depressed after SEPT7 upregulation. To understand the mechanisms by which SEPT7 modulates homeostasis of the actin cytoskeleton, the F‑actin/G‑actin ratio and cofilin expression were determined. The data revealed that the F‑actin/G‑actin ratio and cofilin were reduced, and p‑cofilin increased conversely in cells with SEPT7 overexpression, indicating that SEPT7 reduced glioma cell migration by promoting cofilin phosphorylation and depolymerizing actin. Then, to understand the role of cofilin in SEPT7‑mediated actin dynamic equilibrium and cell migration, cofilin siRNA was transfected into cells. Surprisingly, cell migration and actin polymerization which had been improved by SEPT7 siRNA were significantly reversed, and the accompanying cofilin phosphorylation increased, indicating that cofilin phospho‑regulation played an important role in SEPT7‑mediated cytoskeleton locomotion and glioma cell migration. In conclusion, SEPT7 is involved in glioma cell migration with the assistance of cofilin phospho‑mediated cytoskeleton locomotion.

BACKGROUND/AIMS: Hepatocellular carcinoma is one of the most common cancers worldwide. It has been suggested that microRNAs, a class of small regulatory RNAs, are associated with tumorigenesis by targeting the mRNAs of hundreds of genes that modulate a variety of biological processes, including cellular differentiation, apoptosis, metabolism, and proliferation.
METHODS/RESULTS: we analyzed the expression levels of mir-127 in 33 HCC and non-cancerous tissues using qRT-PCR. MiR-127 is downregulated in 69.7% of HCC tissues compared with adjacent normal tissues, but its expression level is not correlated with the TNM stage, AFP level, or age. In vitro, miR-127 can arrest Huh7 at the G2/M phase and inhibit Huh7 cell proliferation. In an in vivo xenograft model, the overexpression of miR-127 can inhibit Huh7 cell tumorigenicity. The luciferase reporter and western blot results confirm that miR-127 downregulates Sept7 expression by targeting its 3'UTR. Furthermore, the knockdown of Sept7 has the same effect on cell proliferation as the overexpression of miR-127 in Huh7 cells.
CONCLUSION: miR-127 plays a tumor-suppressor role and can serve as a potential diagnostic biomarker for HCC.

MicroRNAs (miRNAs) are small non-coding RNAs of 20-25 nucleotides in length that are capable of modulating gene expression post-transcriptionally. The potential roles of miRNAs in the tumorigenesis of glioblastoma (GBM) have been under intensive studies in the past few years. In the present study, we found a positive correlation between the levels of miR-127-3p and the cell migration and invasion abilities in several human GBM cell lines. We showed that miR-127-3p promoted cell migration and invasion of GBM cells using in vitro cell lines and in vivo mouse models. We identified SEPT7, a known tumor-suppressor gene that has been reported to suppress GBM cell migration and invasion, as a direct target of miR-127-3p. SEPT7 was able to partially abrogate the effect of miR-127-3p on cell migration and invasion. In addition, microarray analysis revealed that miR-127-3p regulated a number of migration and invasion-related genes. Finally, we verified that miR-127-3p affected the remodeling of the actin cytoskeleton mediated by SEPT7 in GBM cells.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by targeting the mRNAs of hundreds of human genes. Variations in miRNA expression levels were shown to be associated with glioma. We have previously found miR-30a-5p overexpression in glioma cell lines and specimens. Bioinformatics analyses predict that several miRNAs, including miR-30a-5p, are involved in the post-transcriptional regulation of SEPT7. SEPT7 is a member of the septin family, which is a highly conserved subfamily of GTPases implicated in exocytosis, apoptosis, synaptogenesis, neurodegeneration and tumorigenesis. Our previous study has also demonstrated that SEPT7 expression is decreased in astrocytic gliomas with different grades and plays a tumor suppressor role. In the present study, we knocked down miR-30a-5p with antisense oligonucleotide (miR-30a-5p AS) in LN229 and SNB19 glioblastoma(GBM) cells, and found that cell growth and invasion were inhibited, while apoptosis was induced. miR-30a-5p AS treated cells showed upregulation of SEPT7 and downregulation of PCNA, cyclin D1, Bcl2, MMP2 and MMP9. In contrast, when miR-30a-5p mimics were transfected into LN229 and SNB19 GBM cells, cell growth and invasion were promoted and the expression of relevant proteins increased. Meanwhile, the effect of miR-30a-5p mimics on glioma cells can be reversed by transfection of SEPT7 construct. Additionaly, miR-30a-5p directly targeting SEPT7 was identified by the reporter gene assay. Our study demonstrates,for the first time, that miR-30a-5p is a bona fide negative regulator of SEPT7 and the oncogenic activity of miR-30a-5p in human gliomas is at least in part through the repression of SEPT7.

Our previous study demonstrated that SEPT7 was downregulated at mRNA level in human gliomas. This study is to further examine the expression of SEPT7 in glioma samples and characterizes its role on cell cycle progression and growth of glioma cells. mRNA and protein expression of SEPT7 were detected by RT-PCR, immunohistochemical staining, and western blot analysis in human glioma specimens and normal brain tissues. A pcDNA3-SEPT7 expression plasmid was constructed and transfected into human glioblastoma cell line U251, and cell proliferation and apoptosis were examined. The growth of established U251 and TJ905 subcutaneous xenograft gliomas was measured in nude mice treated with pcDNA3-SEPT7 and U251 xenograft tumors treated with SEPT7 siRNA. SEPT7 expression is negatively correlated with the increase of glioma grade. Overexpression of SEPT7 is able to inhibit cell proliferation and arrest cell cycle progression in the G0/G1 phase both in vitro and in vivo. Knocking down further the already low endogenous expression of SEPT7 in U251 xenograft tumors with siRNA leads to faster tumor growth compared with control tumors. This study demonstrates that SEPT7 is involved in gliomagenesis and suppresses glioma cell growth.

OBJECTIVES: To explore the role of SEPT7 in glioma cell invasion.
METHODS: SEPT7 was transfected into human glioma cell lines U251 and TJ899, the invasive abilities were evaluated by transwell assay, scratch assay, and 3-D/2-D Matrigel growth. The expression of MMP2/9, MT1-MMP, integrin alpha(v)beta(3), and TIMP1/2 was detected by immunohistochemistry, immunofluorescence, and Western blot analyses. Distribution of alpha-tubulin was examined by laser scanning confocal analysis.
RESULT: After SEPT7 trasfection, cell invasion was inhibited, expression of MMP2/9, MT1-MMP, and integrin alpha(v)beta(3) was decreased, while TIMP1/2 was increased, and alpha-tubulin was redistributed.
CONCLUSION: These results suggest that SEPT7 plays an important role in the glioma cell invasion.

OBJECTIVE: To study the anti-invasion effect of SEPT7 gene on U251MG glioma cells and its possible molecular mechanism.
METHODS: Recombinant adenovirus vector carrying SEPT7 gene (rAd5-SEPT7) was transduced to human glioma cell line U251MG, and empty adenovirus vector was used as control. Tumor invasion was examined by Transwell method and 3 D-Matrigel assay, and tumor cell migration by wound-healing method and 2 D-Matrigel assay. Three major molecular events associated with cell motility and migration, including changes of expression in MMP2, MMP9, MT1-MMP, TIMP1 and TIMP2, the alteration of integrin alpha(v)beta(3) expression, and the structural change of cytoskeleton protein, tubulin-alpha, in U251 cells transduced with rAd5-SEPT7 were studied by Western blotting, immunofluorescence and laser scanning confocal microscope, respectively.
RESULTS: The invasive and migratory capabilities of cells transduced with rAd5-SEPT7 were inhibited. The expression of extracellular matrix metalloproteinases MMP-2, MMP-9, MT1-MMP and integrin alpha(v)beta(3) was significantly decreased, while the expression of matrix metalloproteinase inhibitor TIMP1, TIMP2 was upregulated. Intracellular cytoskeleton protein-tubulin-alpha in U251 cells exhibited prominent morphological changes which including the appearance of distortion and aggregation resulting from redistribution of tubulin-alpha, and this feature of alteration was similar to the tubulin-alpha structure in normal non-tumor cells.
CONCLUSION: SEPT7 gene can inhibit the invasion and migration ability of U251 glioma cells. Its molecular mechanism may include that SEPT7 gene reverses the imbalanced state of MMPs/TIMPs, downregulates the expression of integrin alpha(v)beta(3) and alters the structure of tubulin-alpha of U251MG glioma cells. It is suggested that SEPT7 gene could be a good candidate for gene therapy of gliomas.

OBJECTIVE: To investigate the influence of SEPT7 on biological characters of gliomas cells TJ905.
METHODS: Recombinant SEPT7 constructs was transfected to human glioblastoma cell line TJ905 in which SEPT7 expression is absent. The positive clones were identified by RT-PCR and Western blot analysis. The cell proliferation was determined by MTT assay and flow cytometry, cell apoptosis was detected with Annexin V staining and cell invasion was evaluated by motility in three-dimensional culture. Moreover, the molecules regulating the cell cycle progression were examined by immunofluorescence staining and Western blot analysis.
RESULTS: When SEPT7 was successfully transfected to TJ905 cells, the cell proliferation activity of TJ905 cell was inhibited, the cell cycle was arrested in G0/G1 phase and S phase fraction (SPF) was lowered, the positive regulatory molecules for cell cycle progression including cyclin D1, CDk4, cyclin E and CDk2 were downregulated while the negative modulators including p16 and p21 were upregulated, apoptotic cells were increased and cell invasive ability was attenuated.
CONCLUSIONS: Transfection of SEPT7 construct into the glioma cells TJ905 is able to inhibit the proliferation activity and invasive ability of TJ905 cell and to induce cell apoptosis. These results revealed that SEPT7 exerted the suppressive effect on the glioma cell growth and invasion, and induced apoptosis, and suggested that SEPT7 as a gene of glioma suppressor.

AIM: To explore the effect of androgen receptor (AR) on the expression of the cell cycle-related genes, such as CDKN1A and BTG1, in prostate cancer cell line LNCaP.
METHODS: After AR antagonist flutamide treatment and confirmation of its effect by phase contrast microscope and flow cytometry, the differential expression of the cell cycle-related genes was analyzed by a cDNA microarray. The flutamide treated cells were set as the experimental group and the LNCaP cells as the control. We labeled cDNA probes of the experimental group and control group with Cy5 and Cy3 dyes, respectively, through reverse transcription. Then we hybridized the cDNA probes with cDNA microarrays, which contained 8 126 unique human cDNA sequences and the chip was scanned to get the fluorescent values of Cy5 and Cy3 on each spot. After primary analysis, reverse transcription polymerase chain reaction (RT-PCR) tests were carried out to confirm the results of the chips.
RESULTS: After AR antagonist flutamide treatment, three hundred and twenty-six genes (3.93%) expressed differentially, 97 down-regulated and 219 up-regulated. Among them, eight up-regulated genes might be cell cycle-related, namely CDC10, NRAS, BTG1, Wee1, CLK3, DKFZP564A122, CDKN1A and BTG2. The CDKN1A and BTG1 gene mRNA expression was confirmed to be higher in the experimental group by RT-PCR, while p53 mRNA expression had no significant changes.
CONCLUSION: Flutamide treatment might up-regulate CDKN1A and BTG1 expression in prostate cancer cells. The protein expressions of CDKN1A and BTG1 play an important role in inhibiting the proliferation of cancer cells. CDKN1A has a great impact on the cell cycle of prostate cancer cells and may play a role in the cancer cells in a p53-independent pathway. The prostate cancer cells might affect the cell cycle-related genes by activating AR and thus break the cell cycle control.

Curcumin (diferuloyl methane), the yellow-colored dietary pigment from the rhizomes of turmeric, has been recognized as a chemopreventive agent because of its antitumor, antioxidant and antiproliferative effects. The cytotoxic, apoptotic and gene regulatory effects of both turmeric and curcumin were investigated in the MCF-7 human breast cancer carcinoma cell line and compared with the effects in MCF-10A human mammary epithelial cells. MCF-7 cells were more sensitive to turmeric and curcumin than MCF-10A cells. MCF-10A cells retained comparatively less curcumin in the medium than MCF- 7 cells after 24 h, thereby reducing the cytotoxic effect. Curcumin induced a significantly higher percentage of apoptosis in MCF-7 than MCF-10A cells at all doses. Microarray hybridization of Clonetech apoptotic arrays with labeled first-strand probes of total RNA was performed to identify and characterize the genes regulated by curcumin in tumor cells. Of the 214 apoptosis-associated genes in the array, the expression of 104 genes was altered by curcumin treatment. The gene expression was altered up to 14-fold levels in MCF-7 as compared to only up to 1.5-fold in the MCF-10A cell line by curcumin. Curcumin up-regulated (>3 fold) 22 genes and down-regulated (<3-fold) 17 genes at both 25 microg/ml and 50 microg/ml doses in the MCF-7 cell line. The up-regulated genes include HIAP1, CRAF1, TRAF6, CASP1, CASP2, CASP3, CASP4, HPRT, GADD45, MCL-1, NIP1, BCL2L2, TRAP3, GSTP1, DAXX, PIG11, UBC, PIG3, PCNA, CDC10, JNK1 and RBP2. The down-regulated genes were TRAIL, TNFR, AP13, IGFBP3, SARP3, PKB, IGFBP, CASP7, CASP9, TNFSF6, TRICK2A, CAS, TRAIL-R2, RATS1, hTRIP, TNFb and TNFRSF5. While a dose-dependent gene expression change was noticed in some genes, opposite regulatory effects were induced by different curcumin doses in three apoptotic genes. These results suggest that curcumin induces apoptosis in breast cancer cells by regulation of multiple signaling pathways, indicating its potential use for prevention and treatment of cancer.

To identify genes that are associated with progression of malignant lymphoma, the expression profiles of 18,432 genes were analyzed in diffuse large B-cell lymphomas at early (stages I and II, 6 cases) and advanced stages (stages III and IV, 9 cases) by means of cDNA microarrays. By comparing expression profiles between localized and advanced lymphomas, a number of genes that were differentially expressed were identified: 48 genes with increased expression and 30 genes with reduced expression in advanced-stage diffuse large B-cell lymphomas. Increased expression of MPHOSPH1, RUVBL1, CHN2, PSA and CDC10 genes, and reduced expression of COL1A2, COL4A1, FBLN5, CLECSF6, MIC2, CAV1 and S100A10 genes in the advanced lymphoma group were confirmed by semi-quantitative reverse transcription-PCR. RUVBL1 and PSA expression was further confirmed by real-time quantitative PCR, whose results paralleled the microarray data. The highly expressed genes encode proteins that promote cell proliferation and the genes with reduced expression encode adhesion proteins and target protein for cytotoxic T-lymphocytes. These findings suggested that analysis with cDNA microarrays is a useful approach for identifying genes related to tumor progression and their products could be potential tumor markers or disease-specific targets for anti-tumor therapy.

Diffuse astrocytoma WHO grade II is a well-differentiated, slowly growing tumor that has an inherent tendency to progress to anaplastic astrocytoma (WHO grade III) and, eventually, to glioblastoma (WHO grade IV). Little is known about its molecular basis, except for p53 mutations that are found in >60% of cases. In a search for additional genetic alterations, we carried out gene expression profiling of 11 diffuse astrocytomas using cDNA expression arrays. Expression of six genes (TIMP3, c-myc, EGFR, DR-nm23, nm23-H4, and GDNPF) was detected in 64-100% of diffuse astrocytomas, but not in nontumorous brain tissue. Seven genes (AAD14, SPARC, LRP, PDGFR-alpha, 60S ribosomal protein L5, PTN, and hBAP) were found to be up-regulated more than 2-fold in 20-60% of cases, whereas 11 genes (IFI 9-27, protein kinase CLK, TDGF1, BIN1, GAB1, TYRO3, LDH-A, adducin 3, GUK1, CDC10, and KRT8) were down-regulated to less than 50% of normal levels in 64-100% of cases. Semiquantitative conventional reverse transcription-PCR was performed for 11 genes, 9 of which showed an expression profile similar to that obtained with cDNA expression arrays. Immunohistochemical staining for SPARC showed cytoplasmic immunoreactivity of neoplastic cells in all diffuse astrocytomas analyzed. These results indicate significant changes in gene expression in diffuse astrocytomas, but it remains to be shown which of these are causally related to the transformation of glial cells.

BACKGROUND: The biological behavior of neuroblastomas detected through mass screening (MS, 1 year of age) neuroblastomas have been reported to differ in many studies. To investigate the biological differences between these two groups, we analyzed the differences in mRNA profiles.
MATERIALS AND METHODS: We analyzed the mRNA profiles of MS and MSN neuroblastomas using differential display, and cloned and sequenced the bands differentially expressed between these two groups. Using the RNA analysis by polymerase chain reaction (RNA-PCR) method, the relative amount of mRNA in tumor tissue in each sample was measured. Associations between relative amount of mRNA and clinical and genetic variables related to patient prognosis and the effect of the level of mRNA expression on survival probability were investigated using statistical methods.
RESULTS: Using differential display and RNA-PCR, we found that the mRNA for the human homologue of the yeast cdc10 gene (hCDC10) identified in Saccharomyces cerevisiae was expressed at a higher level in the MS group of patients than in the MSN group of patients (0.554 +/- 0.197 for MS neuroblastoma, n = 24 and 0.244 +/- 0.179 for MSN neuroblastoma, n = 10, P < 0.01), and this difference was suggested to be independent of the histologic subtype of tumor. A high level of hCDC10 mRNA expression in neuroblastomas (relative amount of hCDC10 mRNA > 0.35) was also suggested to be associated with younger age at diagnosis (1 copy, P < 0.05). Patients with neuroblastomas with a high level of hCDC10 mRNA expression were suggested to have a better prognosis than those with a low level of hCDC10 mRNA expression (P < 0.01).
CONCLUSIONS: A high level of hCDC10 mRNA expression in neuroblastomas may be associated with favorable clinical and biological characteristics, and the expression of hCDC10 mRNA in neuroblastomas may affect the clinical and biological characteristics of this type of tumor.

The t(11;17) has been described in patients with acute myeloid leukemia (AML), and the AF17 gene was previously cloned as a fusion partner of the MLL gene in t(11;17)(q23;q21)-AML. We analyzed one patient with de novo AML and one with therapy-related AML with t(11;17)(q23;q25) and identified the AF17q25 gene on chromosome 17q25, a putative septin family gene, fused with MLL. AF17q25 encoded at least three kinds of proteins [type I (568 a.a.), type II (594 a.a.), and type III (574 a.a.)] that contained two kinds of different amino acid sequences at the COOH terminus. The MLL-AF17q25 fusion transcript consisted of type I AF17q25 transcript. The AF17q25 protein is homologous to septin family proteins, including H5, NEDD5, CDC10, and hCDCrel, which is one of the fusion partners of MLL in t(11;22)(q23;q11)-AML. These results suggest that AF17q25 and hCDCrel might define a new septin family particularly involved in the pathogenesis of 11q23-associated leukemia.

Expression of the int-3 locus is activated in mouse mammary tumors as a consequence of insertional mutagenesis by the mouse mammary tumor virus (MMTV). Integration of the MMTV provirus into the int-3 locus promotes the transcription and translation of flanking cellular int-3 sequences sharing significant homology with the intracellular domain of the neurogenic Notch gene of Drosophila, and with the yeast cell cycle regulatory genes cdc10 and SWI6. To determine the in vivo consequences of activated int-3 expression, transgenic mice were generated harboring a genomic tumor DNA fragment consisting of the MMTV LTR and the flanking cellular int-3 sequences. All six int-3 founder transgenic mice and the progeny of one established line exhibited similar dramatic phenotypic abnormalities in tissues in which the transgene was expressed. Focal and often multiple poorly differentiated mammary and salivary adenocarcinomas appeared in the majority of transgenic mice between 2 and 7 months of age. Significantly, mammary glands were arrested in development and were lactation deficient in all female int-3 mice. The salivary glands, glands of the nasal mucosa and maxillary sinus, the extraorbital lacrimal glands, and the Harderian glands of juvenile and adult transgenic mice all contained proliferating immature ductule cells and were incompletely differentiated. In addition, all male int-3 transgenic mice were sterile, apparently the result of severe hyperplasia of the epididymis. These findings demonstrate in vivo that expression of the activated Notch-related int-3 gene causes deregulation of normal developmental controls and hyperproliferation of glandular epithelia.

A gene, bcl-3, is found on chromosome 19 adjacent to the breakpoints in the translocation t(14;19)(q32;q13.1), which occurs in some cases of chronic lymphocytic leukemia. Sequence analysis of the human bcl-3 gene predicts a protein containing seven tandem copies of the SWI6/cdc10 motif. This motif was previously identified in yeast genes that regulate events at the start of the cell cycle and in invertebrate transmembrane proteins involved in cell differentiation pathways. Expression of bcl-3 in normal blood cells increases markedly following mitogenic stimulation, and leukemic cells with the translocation show much greater expression than controls. These results suggest that bcl-3 is a proto-oncogene that may contribute to leukemogenesis when abnormally expressed.