Divergent Matrix-Remodeling Strategies Distinguish Developmental from Neoplastic Mammary Epithelial Cell Invasion Programs.

Metastasizing breast carcinoma cells have been hypothesized to mobilize tissue-invasive activity by co-opting the proteolytic systems employed by normal mammary epithelial cells undergoing branching morphogenesis. However, the critical effectors underlying morphogenesis remain unidentified, and their relationship to breast cancer invasion programs is yet to be established. Here, we identify the membrane-anchored matrix metalloproteinase, Mmp14/MT1-MMP, but not the closely related proteinase Mmp15/MT2-MMP, as the dominant proteolytic effector of both branching morphogenesis and carcinoma cell invasion in vivo. Unexpectedly, however, epithelial cell-specific targeting of Mmp14/MT1-MMP in the normal mammary gland fails to impair branching, whereas deleting the proteinase in carcinoma cells abrogates invasion, preserves matrix architecture, and completely blocks metastasis. By contrast, in the normal mammary gland, extracellular matrix remodeling and morphogenesis are ablated only when Mmp14/MT1-MMP expression is specifically deleted from the periductal stroma. Together, these findings uncover the overlapping but divergent strategies that underlie developmental versus neoplastic matrix remodeling programs.

Expression of mutant p53 proteins implicates a lineage relationship between neural stem cells and malignant astrocytic glioma in a murine model.

Recent studies have identified genes and core pathways that are altered in human glioblastoma. However, the mechanisms by which alterations of these glioblastoma genes singly and cooperatively transform brain cells remain poorly understood. Further, the cell of origin of glioblastoma is largely elusive. By targeting a p53 in-frame deletion mutation to the brain, we show that p53 deficiency provides no significant growth advantage to adult brain cells, but appears to induce pleiotropic accumulation of cooperative oncogenic alterations driving gliomagenesis. Our data show that accumulation of a detectable level of mutant p53 proteins occurs first in neural stem cells in the subventricular zone (SVZ) and that subsequent expansion of mutant p53-expressing Olig2(+) transit-amplifying progenitor-like cells in the SVZ-associated areas initiates glioma formation.

Induction of abnormal proliferation by nonmyelinating schwann cells triggers neurofibroma formation.

Recent evidence suggests that alterations in the self-renewal program of stem/progenitor cells can cause tumorigenesis. By utilizing genetically engineered mouse models of neurofibromatosis type 1 (NF1), we demonstrated that plexiform neurofibroma, the only benign peripheral nerve sheath tumor with potential for malignant transformation, results from Nf1 deficiency in fetal stem/progenitor cells of peripheral nerves. Surprisingly, this did not cause hyperproliferation or tumorigenesis in early postnatal period. Instead, peripheral nerve development appeared largely normal in the absence of Nf1 except for abnormal Remak bundles, the nonmyelinated axon-Schwann cell unit, identified in postnatal mutant nerves. Subsequent degeneration of abnormal Remak bundles was accompanied by initial expansion of nonmyelinating Schwann cells. We suggest abnormally differentiated Remak bundles as a cell of origin for plexiform neurofibroma.

Expression pattern and subcellular localization of five splice isoforms of human PXK.

Phox homology (PX) domains specifically bind to phosphoinositides, and this interaction is crucial for their cellular function. A full-length cDNA of human PX domain containing serine/threonine kinase gene (PXK) that we termed PXK_v1 had previously been cloned. PXK_v1 consists of a S_TKc domain (serine/threonine kinases, catalytic domain), but lacks several residues that are indispensable for intrinsic catalytic activity. Evidence obtained in the present study demonstrated the existence of four other splice isoforms of human PXK in fetal brain, designated as PXK_v2, PXK_v3, PXK_v4 and PXK_v5. The results of RT-PCR indicated that human PXK_v1, PXK_v2 and PXK_v4 transcripts were widely expressed in human adult tissue, except heart tissue. In contrast, PXK_v3 transcripts were only expressed in peripheral blood leukocytes at a low level and PXK_v5 transcripts were not detectable in any of the tissue analyzed. Subcellular localization analysis of EGFP-PXK fusion proteins in COS7 cells indicated that EGFP-PXK_v3 had a different subcellular localization compared to other EGFP-PXK fusion proteins. Mutation analysis of EGFP-PXK_v1 showed that PXK_v1-Tyr56 and Arg92 are essential for subcellular localization of the protein in the cytoplasm.

A novel splice variant of human gene NPL, mainly expressed in human liver, kidney and peripheral blood leukocyte.

From the human fetal brain cDNA library constructed by our lab, a novel variant cDNA of a human gene was successfully cloned and identified. Because the gene has been named N-acetylneuraminate pyruvate lyase (NPL), accordingly we term our splice variant NPL_v2. The cDNA of NPL_v2 has a full-length open reading frame (ORF) from the nucleotide position 320 to 1225 that encodes a protein comprising 301 amino acids. SMART analysis showed that our hypothetical protein has one dihydrodipicolinate synthase (DHDPS) domain. Phosphorylation analysis of the deduced protein show that there are five phosphorylation sites including three "serine" and two "threonine" at the region that are not found in other splice variant. RT-PCR experiment revealed that our splice variant of the gene is mainly expressed in human placenta, liver, kidney, pancreas, spleen, thymus, ovary, small intestine and peripheral blood leukocyte.

A novel splice variant of human XRN2 gene is mainly expressed in blood leukocyte.

The XRN2 gene (XRN2a) is the human homologue of the Saccharomyces cerevisiae RAT1 gene, which encodes a nuclear 5'-->3' exoribonuclease, and is essential for RNA metabolism and cell viability. Xrn2p/Rat1p, product of XRN2/RAT1 gene, functions in the mRNA degradation and processing of rRNAs and small nucleolar RNAs (snoRNAs) in the nucleus. Here we describe the cloning and characterization of a novel splice variant of the human XRN2 gene (XRN2b). The 3271-bp cDNA encodes a putative protein with 907 amino acid residues, which shares high homology with mouse DHM1 protein. RT-PCR analysis showed that XRN2b was mainly expressed in blood leukocyte tissue, while XRN2a was detected in several human tissues and in human tumor tissues.

Molecular cloning and characterization of a novel human C4orf13 gene, tentatively a member of the sodium bile acid cotransporter family.

By large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a novel human cDNA (C4orf13). This cDNA is 2706 bp in length, encoding a 340-amino-acid polypeptide that contains a typical SBF (sodium bile acid cotransporter family) domain and ten possible transmembrane segments. The putative protein C4orf13 shows high similarity with its orthologs in Mus musculus and Xenopus laevis. Human C4orf13 is mapped to chromosome 4q31.2 and contains 12 exons. RT-PCR analysis shows that human C4orf13 is widely expressed in human tissues, and the expression levels in liver and lung are relatively high, expression levels in placenta, kidney, spleen, and thymus are moderate, low levels of expression are detected in heart, prostate, and testis.

Cloning and characterization of a novel RNA polymerase II C-terminal domain phosphatase.

Reversible phosphorylation of RNA polymerase (RNAP) II's largest subunit C-terminal domain (CTD) is a key event during mRNA metabolism. The CTD phosphatase, FCP1, catalyzes the dephosphorylation of RNAP II and is thought to play a major role in polymerase recycling. In this study, we isolated a novel phosphatase gene by large-scale sequencing analysis of a human fetal brain cDNA library. Its cDNA is 2215 bp in length, encoding a 318-amino acid polypeptide that contains a ubiquitin-like domain and a CTD phosphatase domain. Therefore, it was termed ubiquitin-like domain containing CTD phosphatase 1 (UBLCP1). Reverse transcription PCR (RT-PCR) revealed that UBLCP1 was expressed with relatively lower levels in most adult normal tissues and higher levels in fast growing or tumor tissues. Transient transfection experiment suggested that UBLCP1 was localized in the nucleus of COS-7 cells. Significantly, UBLCP1 could dephosphorylate GST-CTD in vitro. Accordingly, UBLCP1 may play a role in the regulation of phosphorylation state of RNA polymerase II C-terminal domain.

Molecular cloning and characterization of a novel human gene containing 4 ankyrin repeat domains.

Ankyrin repeat, one of the most important protein motifs, plays a wide variety of roles in protein-protein interactions and in the signal pathways. Via large-scale sequencing, a novel 941-bp gene was isolated from an 18-week old human fetal brain cDNA library. It encodes a putative protein of 158 amino acid residues with four conserved ankyrin repeat domains. It displays a high degree of homology with rat low-density lipoprotein receptor-related protein 2-binding protein (Lrp2bp), and was therefore was named hLrp2bp (human Lrp2bp). The hLrp2bp gene was located in chromosome 4q35 and the conserved ankyrin repeat domains were located between amino acid residues 10 and 116. RT-PCR revealed that hLrp2bp was mainly expressed in the human testis, small intestine, colon and blood leukocytes, and in human pancreatic adenocarcinoma cells. A HEK293 cell was transfected with the ORF of hLrp2bp, and analyses showed that the protein was distributed both in the cytoplasm and nucleus.

Cloning and analysis of human Apg16L.

Autophagy is an intracellular bulk degradation system, which delivers cytoplasmic components to the lysosome/vacuole. In yeast and mammalian cells, the Apg12-Apg5 conjugate, together with Apg16, form a multimeric complex, which plays an essential role in autopihageosome formation. By large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a cDNA encoding a putative protein with 607 amino acid residues, which shows 90% identity and 93% similarity to mouse Apg16L. This protein, designated human Apg16L, contains a coiled-coil domain and a motif with seven WD repeats, which are also shared by mouse Apg16L. Database searching revealed that Apg16L is mapped to chromosome 2q37.1 and there exist at least four splice variants.

Characterization of a cDNA encoding a protein with limited similarity to beta1, 3-N-acetylglucosaminyltransferase.

Glycosyltransferases constitute a large group of enzymes that are involved in a wide range of functions in all living organisms. By large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a novel human putative glycosyltransferase gene named beta3GnTL1. Its cDNA is 1372 base pair in length, encoding a predicted protein with 361 amino acid residues. The human beta3GnTL1 is located to chromosome 17q25.3 by comparison of its cDNA with human gemome database. RT-PCR result shows the beta3GnTL1 is expressed at various levels in most of tissues examined.

Molecular cloning and characterization of SGT1.2, a novel splice variant of Homo sapiens SGT1.

SGT1.2, a novel splice variant of Homo sapiens SGT1 was isolated from a human fetal brain cDNA library. This cDNA is 1404 bp and contains an open reading frame from 68 to 1165 encoding a putative protein of 365 amino acids (SGT1.2) that share 90% identities to Homo sapiens SGT1, suppressor of G2 allele of SKP1 at protein level. RPS-BLAST searching revealed that SGT1.2 have a TPR domain, a p23 domain, a SGS domain and a CS domain. According to the search of the human genome database, SGT1.2 was mapped to human chromosome 13q14.13. Reversed transcription-polymerase chain reaction analysis indicated that it widely expressed in human adult tissues.

Cloning and identification of a novel human gene PDLIM5, a homolog of AD-associated neuronal thread protein (AD7c-NTP).

A novel human gene cDNA was successfully cloned from the human fetal brain cDNA library constructed by our lab, and this gene was termed PDLIM5 after acquiring the agreement of HUGO. BLASTX searching revealed that the hypothetical protein is a homolog of AD-associated neuronal thread protein (AD7c-NTP), which is over-expressed in Alzheimer disease (AD) beginning early in the course of disease, and over-expression of the AD7c-NTP gene would cause neuritic sprouting and cell death. SMART analysis showed that both our predicted protein and AD7c-NTP comprise BCL domain (only contains BH1 and BH2 regions). RT-PCR experiment revealed that the expression level of PDLIM5 in brain, skeletal muscle, prostate, colon and leukocyte is obviously higher than that in other tissues.

Cloning, characterization and expression of CDK5RAP1_v3 and CDK5RAP1_v4, two novel splice variants of human CDK5RAP1.

Two novel splice variants of CDK5RAP1, named CDK5RAP1_v3 and CDK5RAP1_v4, were isolated through the large-scale sequencing analysis of a human fetal brain cDNA library. The CDK5RAP1_v3 and CDK5RAP1_v4 cDNAs are 1923bp and 1792bp in length, respectively. Sequence analysis revealed that CDK5RAP1_v4 lacked 1 exon, which was present in CDK5RAP1_v3, with the result that these cDNAs encoded different putative proteins. The deduced proteins were 574 amino acids (designated as CDK5RAP1_v3) and 426 amino acids (CDK5RAP1_v4) in length, and shared the 420 N-terminal amino acids. RT-PCR analysis showed that human CDK5RAP1_v3 was widely expressed in human tissues. The expression level of CDK5RAP1_v3 was relatively high in placenta and lung, whereas low levels of expression were detected in heart, brain, liver, skeletal muscle, pancreas, spleen, thymus, small intestine and peripheral blood leukocytes. In contrast, human CDK5RAP1_v4 was mainly expressed in brain, placenta and testis.

A novel splice variant of human gene GDPD1 is mainly expressed in human ovary and small intestine.

Glycerophosphodiester phosphodiesterase (GDPD) is a glycerol metabolizing enzyme that has been previously identified in Escherichia coli, Haemophilus influenzae, Bacillus subtilis and Borrelia hermsii. It has been also reported that there is putative protein containing GDPD domain in Mus musculus, but not in Homo sapiens. SMART analysis showed that our predicted protein and three others of the same gene also comprise this domain. Because the gene has not been named, we termed it GDPD1 and accordingly termed our splice variant GDPD1_v1 after acquiring the agreement of HUGO. RT-PCR experiment revealed that our splice variant of the gene is mainly expressed in human ovary and small intestine, basically conforming to the result of serial analysis of gene expression.

Molecular cloning and characterization of a novel human putative transmembrane protein homologous to mouse sideroflexin associated with sideroblastic anemia.

Sideroflexin1 (Sfxn1), the prototype of a novel family of evolutionarily conserved proteins present in eukaryotes, has been found mutated in mice with siderocytic anemia. It is speculated that this protein facilitates the transport of a component required for iron utilization into mitochondrial. During the large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a cDNA encoding a novel sideroflexin protein (SFXN4), which showed 59% identity and 71% similarity to mouse sideroflexin4. According to the search of the human genome database, SFXN4 gene is mapped to chromosome 10q25-26 and spans more than 24.7kb of the genomic DNA. It is 1428 base pair in length and the putative protein contains 305 amino acids with a conserved predicted five-transmembrane-domains structure. RT-PCR result shows that the SFXN4 gene is expressed in many tissues.