Silencing of miR-148a in cancer-associated fibroblasts results in WNT10B-mediated stimulation of tumor cell motility.

The tumor microenvironment has an important role in cancer progression. Here we show that miR-148a is downregulated in 15 out of 16 samples (94%) of cancer-associated fibroblasts (CAFs) compared with matched normal tissue fibroblasts (NFs) established from patients with endometrial cancer. Laser-capture microdissection of stromal cells from normal tissue and endometrial cancer confirmed this observation. Treatment of cells with 5-aza-deoxycytidine stimulated the expression of miR-148a in the majority of CAFs implicating DNA methylation in the regulation of miR-148a expression. Investigation of miR-148a function in fibroblasts demonstrated that conditioned media (CM) from CAFs overexpressing miR-148a significantly impaired the migration of five endometrial cancer cell lines without affecting their growth rates in co-culture experiments. Among predicted miR-148a target genes are two WNT family members, WNT1 and WNT10B. Activation of the WNT/ß-catenin pathway in CAFs was confirmed by microarray analysis of gene expression and increased activity of the SuperTOPFlash luciferase reporter. We found elevated levels of WNT10B protein in CAFs and its level decreased when miR-148a was re-introduced by lentiviral infection. The 3'-UTR of WNT10B, cloned downstream of luciferase cDNA, suppressed luciferase activity when co-expressed with miR-148a indicating that WNT10B is a direct target of miR-148a. In contrast to the effect of miR-148a, WNT10B stimulated migration of endometrial cancer cell lines. Our findings have defined a molecular mechanism in the tumor microenvironment that is a novel target for cancer therapy.

BRCA1 is a selective co-activator of 14-3-3 sigma gene transcription in mouse embryonic stem cells.

BRCA1 gene is a tumor suppressor for breast and ovarian cancers with the putative role in DNA repair and transcription. To characterize the role of BRCA1 in transcriptional regulation, we analyzed gene expression profiles of mouse embryonic stem cells deficient in BRCA1 using microarray technology. We found that loss of BRCA1 correlated with decreased expression of several groups of genes including stress response genes, cytoskeleton genes, and genes involved in protein synthesis and degradation. Previous study showed that BRCA1 is a transcriptional co-activator of p53 protein; however the majority of p53 target genes remained at the same expression levels in BRCA1 knockout cells as in the wild type cells. The only p53 target gene down-regulated with the loss of BRCA1 was 14-3-3 sigma, a major G(2)/M checkpoint control gene. Similar to cells with decreased 14-3-3 sigma activity, BRCA1-deficient cells were unable to sustain G(2)/M growth arrest after exposure to ionizing radiation. We find that BRCA1 induction of 14-3-3 sigma requires the presence of wild type p53 and can be regulated by a minimal p53 response element.

Construction and characterization of recombinant adenoviruses expressing human BRCA1 or murine Brca1 genes.

Recombinant adenoviruses expressing human BRCA1 (AdBRCA1), murine Brca1 (AdBrca1), three clinically relevant human mutant BRCA1 proteins (t340, C61G, and 1853Stop), or a murine Brca1 C-terminal deletion mutant were constructed and evaluated in vitro. These recombinants were capable of transducing high-level transgene expression to a wide variety of cell lines in vitro. Three independent methods were utilized to monitor cell growth following transduction with these recombinants. High-level expression of either the human or mouse wild-type BRCA1 protein was incompatible with maximal levels of cell growth. AdBRCA1 transduction inhibited the outgrowth of several human breast and ovarian cell lines in colony formation assays. Flow cytometric analysis revealed an accumulation of the transduced cells in the G0/G1 phase of the cell cycle. This BRCA1-mediated accumulation of cells in G0/G1 was accompanied by an increase in the cellular level of hypophosphorylated pRB. Ad mutant BRCA1 t340, C61G, and 1853Stop viruses were impaired, to varying degrees, in their ability to transduce a growth-arrested state to the target cells. Using these same three criteria, overexpression of murine Brca1 by AdBrca1 was also capable of transducing a growth-arrested state to human cells. Deletion of the C-terminus of Brca1 diminished this activity. This panel of adenoviruses may be useful reagents as part of an approach to understand the function of BRCA1/Brca1 in normal breast and ovary and help to define the tumor suppressor defect (s) conferred by clinical BRCA1 mutations in breast and ovarian cell tumorigenesis.

BRCA1-associated growth arrest is RB-dependent.

BRCA1 is a susceptibility gene for breast and ovarian cancer with growth-inhibitory activity for which the mechanism of action remains unclear. When introduced into cells, BRCA1 inhibits growth of some but not all cell lines. In an attempt to uncover the mechanism of growth suppression by BRCA1, we examined a panel of cell lines for their ability to reduce colony outgrowth in response to BRCA1 overexpression. Of all variables tested, only those cells with wild-type pRb were sensitive to BRCA1-induced growth suppression. In cells with an intact rb gene, inactivation of pRb by HPV E7 abrogates the growth arrest imposed by BRCA1. In accordance with these observations, we found that BRCA1 could not suppress BrdUrd uptake in primary fibroblasts from rb-/- mice and exhibited an intermediate ability to inhibit DNA synthesis in rb+/- as compared with rb+/+ cells. We further found that the BRCA1 protein complexes with the hypophosphorylated form of pRb. This binding is localized to amino acids 304-394 of BRCA1 protein and requires the ABC domain of pRb. In-frame deletion of BRCA1 fragment involved in interaction with pRb completely abolished the growth-suppressive property of BRCA1. Although it has been reported that BRCA1 interacts with p53, we find the p53 status did not affect the ability of BRCA1 to suppress colony formation. Our data suggest that the growth suppressor function of BRCA1 depends, at least in part, on Rb.

BRCA1 protein level is not affected by peptide growth factors in MCF10A cell line.

The breast cancer susceptibility gene (BRCA1) has been identified as a putative tumor suppressor on chromosome 17. We raised antibody against Ring-finger domain of BRCA1. The antibody recognizes a specific BRCA1 protein doublet of about 220 kD. The majority of BRCA1 protein is localized to the nuclear fraction of untreated MCF10A cells. Though BRCA1 is thought to be a growth suppressor gene, no change in BRCA1 protein level was found when MCF10A cells were arrested by growth factor deprivation or stimulation of cell proliferation by re-addition of growth factors. Furthermore the subcellular localization of the BRCA1 protein does not change throughout the cell cycle. These results suggest that BRCA1 may not be directly involved in the regulation of the cell cycle of breast cancer cell line.

Both p16 and p21 families of cyclin-dependent kinase (CDK) inhibitors block the phosphorylation of cyclin-dependent kinases by the CDK-activating kinase.

Phosphorylation of cyclin-dependent kinases (CDKs) by the CDK-activating kinase is required for the activation of CDK enzymes. Members of two families of CDK inhibitors, p16/p18 and p21/p27, become physically associated with and inhibit the activity of CDKs in response to a variety of growth-modulating signals. Here, we show that the representative members of both families of CDK inhibitors, p21waf1,cip1, p27kip1, and p18, can prevent the phosphorylation of their CDK partners, CDK2 and CDK6, by CDK-activating kinase. No direct interaction between CDK-activating kinase and the CDK inhibitors could be detected, suggesting that binding of these CDK inhibitors to CDK subunits renders CDK inaccessible to the CDK-activating kinase phosphorylation. These findings suggest that a general mechanism of CDK inhibitor function is to block the phosphorylation of CDK enzymes by CDK-activating kinase.

Signalling properties of FLT4, a proteolytically processed receptor tyrosine kinase related to two VEGF receptors.

The FLT4, FLT1 and KDR/FLK1 genes encode structurally similar endothelial cell receptor tyrosine kinases. Recently it has been shown that the FLT1 and KDR/FLK-1 proteins function as high-affinity receptors for vascular endothelial growth factor (VEGF). Here we show that FLT4 does not act as a receptor for VEGF, as VEGF did not show specific binding to the FLT4 tyrosine kinase or induce its autophosphorylation. Also, FLT4 did not interact with KDR in response to VEGF. However, when fused with the ligand binding domain of the colony stimulating factor-1 receptor (CSF-1R), the FLT4 tyrosine kinase was specifically activated by CSF-1. The activated FLT4 tyrosine kinase domain was found to interact with the Src homology 2 domains of the SHC and GRB2 adaptor proteins in vitro and with SHC in cells. CSF-1 stimulation of the CSF-1R/FLT4 receptor chimera induced thymidine incorporation in serum-starved NIH3T3 fibroblasts, but not in porcine aortic or murine lung capillary endothelial cells, although tyrosyl phosphorylation of the receptor and SHC occurred in these cells as well. These results suggest that the endothelial cell FLT4 receptor tyrosine kinase transmits signals for an as yet unidentified growth factor.

The related FLT4, FLT1, and KDR receptor tyrosine kinases show distinct expression patterns in human fetal endothelial cells.

The growth factor receptors expressed on endothelial cells are of special interest because of their potential to program endothelial cell growth and differentiation during development and neovascularization in various pathological states, such as wound healing and angiogenesis associated with tumorigenesis. Vascular endothelial growth factor ([VEGF] also known as vascular permeability factor) is a potent mitogen and permeability factor, which has been suggested to play a role in embryonic and tumor angiogenesis. The newly cloned FLT4 receptor tyrosine kinase gene encodes a protein related to the VEGF receptors FLT1 and KDR/FLK-1. We have here studied the expression of FLT4 and the other two members of this receptor family in human fetal tissues by Northern and in situ hybridization. These results were also compared with the sites of expression of VEGF and the related placenta growth factor (PlGF). Our results reveal FLT4 mRNA expression in vascular endothelial cells in developing vessels of several organs. A comparison of FLT4, FLT1 and KDR/FLK-1 receptor mRNA signals shows overlapping, but distinct expression patterns in the tissues studied. Certain endothelia lack one or two of the three receptor mRNAs. These data suggest that the receptor tyrosine kinases encoded by the FLT gene family may have distinct functions in the regulation of the growth/differentiation of blood vessels.

Two human FLT4 receptor tyrosine kinase isoforms with distinct carboxy terminal tails are produced by alternative processing of primary transcripts.

FLT4 is a recently cloned gene encoding a transmembrane tyrosine kinase related to the FLT1 and KDR/FLK1 vascular endothelial growth factor receptors. We have previously shown that FLT4 is expressed as transcripts of 4.5 and 5.8 kb in several human fetal and adult tissues. Here we show that these transcripts encode two polypeptides, FLT4s (short) and FLT41 (long), which are proteolytically processed in transfected cells and leukemia cells and which have different carboxy terminal tails. The 3' coding region of the 5.8 kb mRNA was found to be 65 codons longer than that of the the 4.5 kb mRNA. Analysis of the genomic structure of the region encoding the two carboxy termini revealed that the two transcripts are generated by alternative polyadenylation and subsequent alternative splicing during RNA processing. Our findings thus show regulation of FLT4 structure in the carboxy terminal tail considered important for receptor function. The significance of the two forms may relate to the role of additional potential autophosphorylation sites in the FLT4 long form.

FLT4 receptor tyrosine kinase gene mapping to chromosome band 5q35 in relation to the t(2;5), t(5;6), and t(3;5) translocations.

FLT4 is a recently cloned receptor tyrosine kinase cDNA, which is characterized by seven immunoglobulin-like loops in its extracellular domain. We have previously mapped the FLT4 gene to chromosome segment 5q33-qter using somatic cell hybrids. Here we have refined the localization to band 5q35 by fluorescence in situ hybridization and show that the gene is translocated to chromosomes 2 and 6 in the t(2;5)(p23;q35) and t(5;6)(q35;p21) translocations, respectively, of Ki-I-positive lymphomas, as well as to chromosome 3 in the t(3;5)(q25.1;q34) translocation, which is occasionally found in myelodysplastic syndromes and acute myeloid leukemia. No evidence was obtained for a rearrangement or deregulation of the translocated FLT4 gene. We further show that abundant FLT4 mRNA expression occurs only in erythroid and megakaryoblastoid cell lines among nine leukemia cell lines studied.

Two alternative mRNAs coding for the angiogenic factor, placenta growth factor (PlGF), are transcribed from a single gene of chromosome 14.

We have previously reported on the identification of a cDNA (placenta growth factor, PlGF) coding for a novel angiogenic factor expressed in placental tissue that is similar to vascular permeability factor/vascular endothelial growth factor (VPF/VEGF). Biochemical and functional characterization of PlGF derived from transfected COS-1 cells revealed that it is a glycosylated dimeric secreted protein able to stimulate endothelial cell growth in vitro. Here, we report the isolation and characterization of the PlGF gene located on chromosome 14. At least two different mRNAs are produced from this single-copy gene in different cell lines and tissues. Sequence comparison of the polypeptides encoded by the two different isolated cDNAs indicates that they are identical except for the insertion of a highly basic 21 amino acid stretch at the carboxyl end of the protein. RNA expression analysis of several tissues, tumors and cell lines indicates differential distribution of the two PlGF mRNAs. Finally, preliminary results indicate that the PIGF gene has been conserved in evolution, since the human PlGF cDNA hybridizes to sequences present in the genomic DNA of Drosophila, Xenopus, chicken and mouse.

FLT4 receptor tyrosine kinase contains seven immunoglobulin-like loops and is expressed in multiple human tissues and cell lines.

The fms-like tyrosine kinase 4 (FLT4) complementary DNA was cloned from a human HEL erythroleukemia cell library by polymerase chain reaction-amplification. We previously reported a partial sequence of FLT4 and showed that the FLT4 gene maps to chromosomal region 5q33-qter (O. Aprelikova, K. Pajusola, J. Partanen, E. Armstrong, R. Alitalo, S. Bailey, J. McMahon, J. Wasmuth, K. Huebner, and K. Alitalo, Cancer Res., 52: 746-748, 1992). Here we present the full-length sequence of the predicted FLT4 protein. The extracellular domain of FLT4 consists of 7 immunoglobulin-like loops, including 12 potential glycosylation sites. On the basis of structural similarities FLT4 and the previously known FLT1 and kinase insert domain-containing receptor tyrosine kinase/fetal liver kinase 1 (KDR/FLK1) receptors constitute a subfamily of class III tyrosine kinases. FLT4 was expressed as 5.8- and 4.5-kilobase mRNAs which were found to differ in their 3' sequences and to be differentially expressed in the HEL and DAMI leukemia cells. Interestingly, a Wilms' tumor cell line, a retinoblastoma cell line, and a nondifferentiated teratocarcinoma cell line expressed FLT4, whereas differentiated teratocarcinoma cells were negative. Most fetal tissues also expressed the FLT4 mRNA, with spleen, brain intermediate zone, and lung showing the highest levels. In in situ hybridization the FLT4 autoradiographic grains decorated bronchial epithelial cells of fetal lung. No evidence was obtained for the expression of FLT4 in the endothelial cells of blood vessels.

A radiation hybrid map of 18 growth factor, growth factor receptor, hormone receptor, or neurotransmitter receptor genes on the distal region of the long arm of chromosome 5.

The distal portion of the long arm of human chromosome 5 contains an impressive number of genes encoding growth factors, growth factor receptors, and hormone/neurotransmitter receptors. The order of and relative distance between 18 of these genes was determined by radiation hybrid mapping. There is only a single gap in a contiguous radiation map from 5q22-5q35. For this set of radiation hybrids, one map unit (centiray) corresponds to 20-50 kb of DNA. Close physical proximity for several pairs of loci was predicted by the map. Two sets of these were found to be contained in single YAC clones. The physical map produced by radiation hybrid mapping should prove useful in efforts to identify four disease genes that have been assigned to distal 5q by linkage studies.

FLT4, a novel class III receptor tyrosine kinase in chromosome 5q33-qter.

The receptors for at least two hematopoietic growth factors, namely the stem cell factor and colony-stimulating factor 1, belong to class III receptor tyrosine kinases. Here we describe cloning of a partial complementary DNA for FLT4, an additional member of this gene family from human leukemia cells. The FLT4 tyrosine kinase domain is 79% homologous with the previously cloned FLT1 (M. Shibuya et al., Oncogene, 5: 519-524, 1990) tyrosine kinase and maps to the chromosomal region 5q33-qter. We have found FLT4 expression in human placenta, lung, heart, and kidney, whereas the pancreas and brain appeared to contain very little if any FLT4 RNA. The results suggest that FLT4 functions in multiple adult tissues.

[The levels of DNA repair in the cells of diploid and tetraploid cell cultures]. / Issledovanie urovnei reparatsii DNK v kletkakh diploidnykh i tetraploidnykh kletochnykh kul'tur.

Levels of unscheduled DNA synthesis after UV-irradiation were investigated in addition to the activities of DNA-repair enzyme uracil-DNA glycosilase in cells of both diploid (XC-D) and tetraploid (XC-T) cell cultures. It was shown that repair levels were increasing directly and proportionally to the cell ploidy.

[Genetic transformation of somatic cells. XIV. Expression of gene coding for the human hepatitis B virus surface antigen in mammalian cells]. / Geneticheskaia transformatsiia somaticheskikh kletok. XIV. Ekspressiia gena, kodiruiushchego poverkhnostnyi antigen virusa gepatita B cheloveka, v kletkakh mlekopitaiushchikh.

A set of recombinant plasmids with a gene encoding surface antigen of hepatitis B virus (HBsAg) is constructed. The plasmids were transfected by DEAE-dextran method into different lines of cultured cells and transient expression of the HBsAg gene was studied. The results indicate that: transcriptional enhancer of hepatitis B virus situated downstream from HBsAg gene is active in green monkey kidney cells (CVI), promoter of 5 LTR of bovine leukemia virus is trans-activated in the goat or calf cells infected with BLV. The results are discussed in the light of hypothesis on the role of transcriptional enhancers in determination of tissue-specificity of hepatitis B virus.

[Transformation of mice by a prokaryotic gene for dihydrofolate reductase]. / Transformatsiia myshei prokarioticheskim genom digidrofolatreduktazy.

In mice obtained after microinjection into the male pronucleus of fertilized eggs of the plasmid, containing the bacterial gene of dihydrofolate reductase (DHFR), under the control of the early promotor of the simian virus 40 (SV40), an integration of the foreign DNA into the mouse genome is found. About 30% of the treated animals contain the integrated plasmid DNA sequences, i.e. are transgenic. In 2 of 7 mice, containing the introduced plasmid in their genome, the methotrexate-resistant DHFR activity is found in the kidney and spleen, which may be due to the expression of gene DHFR. The plasmid DNA sequences and the ability to synthesise the methotrexate-resistant enzyme DHFR are transmitted to the next generation of mice.