Molecular genetic approaches to the study of cellular senescence.

Normal cells in culture exhibit limited division potential, which is used as a model for cellular aging. In contrast, tumor-derived, carcinogen- or virus-transformed cells are capable of dividing indefinitely (immortal). Fusion of normal with immortal human cells yielded hybrids having limited life span, indicating that cellular senescence is a dominant phenotype and that immortality is recessive. Fusions of various immortal human cell lines with each other led to the identification of four complementation groups for indefinite division. In order to identify the chromosomes and genes involved in growth regulation, that had been modified in immortal cells, we used the technique of microcell fusion to introduce either a normal human chromosome 11 or 4 into cell lines representative of the different complementation groups. Chromosome 11 had no effect on the in vitro life span of the different immortal human tumor lines. However, when a normal human chromosome 4 was introduced into cell lines assigned to complementation group B, the cells lost the immortal phenotype. No effect on the proliferation potential of cell lines representative of the other complementation groups was observed. These results suggest that a gene(s) on human chromosome 4 has been modified in immortal cell lines assigned to complementation group B, to allow escape from senescence. They also provide evidence for a genetic basis for cellular aging.     

Current status of chromosomal abnormalities in mouse embryonic stem cell lines used in Japan

We performed chromosomal analysis on 540 mouse embryonic stem (ES) cell lines obtained during 2001 to 2004 from 20 institutions in Japan. Overall, 66.5% of the ES cell lines showed normal chromosomal numbers, but 15.9%, 9.1%, and 2.8% showed modal chromosomal numbers of 41, 42, and 39, respectively. When we karyotyped 88 ES cell lines selected arbitrarily from the 540 lines, 53 (60.2%) showed normal diploid karyotypes; the sex chromosome constitution of 52 lines was XY, with the remaining 1 being XX. Among 35 ES cell lines showing abnormal karyotypes, trisomy of chromosome 8 (41, XY, +8) was dominant (51.4%), 14.3% had trisomy 8 with loss of one sex chromosome (40, XO, +8), and 11.4% had trisomy 8 together with trisomy 11 (42, XY, +8, +11). Karyotypic abnormalities including trisomy 8 and trisomy 11 occurred in 88.6% and 17.1% of ES cell lines, respectively. The XO sex chromosome constitution was observed in 25.7% of all abnormal ES cell lines. Of the 88 selected ES cell lines, 60 lines were established from strain 129 animals, 17 from F1 progeny of C57BL/6J x CBA (called TT2 in this study), and 11 from C57BL/6J mice. Normal diploid karyotypes were observed in 58.3% of lines derived from 129, 58.8% of those from TT2, and 72.7% of C57BL/6J. The relatively high incidence of abnormalities in chromosomal number and karyotype in ES cell lines used in Japan suggests the importance of chromosomal analysis of ES cells for successful establishment of new animal models through germline transmission.     

Altered placental morphology associated with murine trisomy 16 and murine trisomy 19

The morphology of placentas from trisomy 16 and trisomy 19 mouse conceptuses aged 12 to 18 gestational days was studied at the light microscopic level. Comparisons were made with placentas from normal littermate animals. Trisomy 16 placentas showed marked changes from normal: 1) the junctional zone showed little indication of normal morphologic differentiation throughout gestation; 2) clusters of germinal trophoblast cells persisted in the labyrinth throughout gestation, whereas these cells disappeared by gestational day 16 in the normal littermate placentas; 3) the labyrinth was reduced in size in the trisomic placentas, and the differentiation of the interhemal membranes was delayed. The size of the labyrinths from trisomy 19 placentas appeared to be decreased, but otherwise the placentas appeared to have normal morphology. These observations and others from the literature show that placental development is affected by the presence of a trisomic genome, and that different trisomies influence the development of the placenta differently. For trisomy 16, we propose that the striking changes of the junctional zone may be associated with the trisomy 16-related gene dosage effect for alpha- and beta-interferon cell surface receptors. Because of the homology for this and other genes on mouse chromosome 16 with genes on human chromosome 21, findings related to the altered development of the trisomy 16 mouse may be relevant to understanding some of the phenotypic variations associated with human trisomy 21, the Down syndrome.     

Endothelial cells are activated by angiopoeitin-1 gene transfer and produce coordinated sprouting in vitro and arteriogenesis in vivo

RATIONAL AND OBJECTIVES: Activation of fully differentiated vascular cells using angiogenic genes can lead to phenotypic changes resulting in formation of new blood vessels. We tested whether Ang-1 gene transfer to endothelial cells (EC) activates these cells. METHODS AND RESULTS: EC and SMC were transduced using retroviral or adenoviral vectors to produce Ang-1 or vascular endothelial growth factor (VEGF). EC Tie-2 receptor was phosphorilated by autologous secretion of Ang-1. Transduced EC and SMC sprouting capacity was tested using collagen embedded spheroids assay and capacity to produce arteriogenesis was tested in a hind limb model of ischemia. EC expressing Ang-1 in the presence of SMC expressing VEGF exhibited high levels of sprouting of the two cell types. Flow and numbers of arteries were increased after transduced cells implantation in vivo. CONCLUSIONS: Autologous secretion of Ang-1 by transduced EC resulted in Tie-2 activation and in the presence of SMC expressing VEGF resulted in coordinated sprouting in vitro and increase in flow and number of arteries in vivo.     

Chinese hamster x American mink somatic cell hybrids: characterization of a clone panel and assignment of the mink genes for malate dehydrogenase,NADP-1 and malate dehydrogenase,NAD-1

Summary Chinese hamster x American mink somatic cell hybrids were obtained and examined for chromosome content and expression of mink malate dehydrogenase, NADP (MOD-1; EC 1.1.1.40), malate dehydrogenase, NAD (MOR-1; EC 1.1.1.37), glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) and hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8). All the hybrid clones examined were found to segregate mink chromosomes. A clone panel containing 25 clones was set up. The possibilities and limitations of this panel for mink gene mapping are analysed. Using this panel, it is feasible to rapidly map genes located on chromosomes 1–13 and to provisionally assign genes located on chromosome 14 and the X. Based on the data obtained, the genes for MOD-1 and MOR-1 were firmly assigned to mink chromosomes 1 and 11, respectively, and the genes for G6PD and HPRT were provisionally assigned to the X.     

Partial proximal trisomy 10q syndrome: a new case

We report a case of partial proximal trisomy of the long arm of chromosome 10 confirmed by fluorescence in situ hibridization (FISH) performed with whole chromosome 10 specific painting and specific yac clones. The phenotypic findings, compared to those found in other published cases with the same karyotype, support the recognition of a distinctive partial proximal trisomy 10q syndrome (10q11-->q22).     

Detection of DNA copy number abnormality by microarray expression analysis

Gene copy-number abnormalities (CNAs) are characteristic of solid tumors and are found in association with developmental abnormalities and/or mental retardation. The ultimate impact of CNAs is exerted by the altered expression of encoded genes. We have utilized high-density oligonucleotide arrays from Affymetrix to identify DNA CNAs via their impact on mRNA expression levels. In these studies, we have used three different trisomic cell lines (trisomy 9, trisomy 18, trisomy 21) as models of CNAs and have compared mRNA expression in those trisomic cells with that observed in diploid cell lines of matched tissue origin. Our data clearly show that genes from CNA chromosome regions are substantially over-represented (P<0.000001 by chi-square analysis) in the differentially expressed subset from comparisons of all three trisomic cell lines with normal matching cells. In addition, we have been able to detect the origin of the duplication by a statistical scan for over-expressed genes. These data show that microarray detection of differential mRNA expression can be used to identify significant DNA CNAs.     

Chromosomal rearrangements and their effects in spontaneous immortalization and transformation of rat embryo cells in vitro

G-banding analysis of LRec-1 and LRec-3, spontaneously immortalized cell lines from rat embryo fibroblast, revealed diploid karyotypes with specific clonal structural rearrangements of chromosomes 7 and 19 - del(7)(q11.2q22.1), t(7;19)(q11.1;q12) in malignant stage. Both clonal abnormalities of chromosomes 7 and 19 were also revealed in LRec-1k clone and LRec-1 sf cell line. Previous study of LRec-1 and LRec-3 cells showed the presence of karyotypes with pseudodiploid modal chromosome number, partial trisomy of chromosome 7 and same clonal structural rearrangements of chromosomes 7 and 19 in immortalized stage. In malignant stage, the trisomy 6 and new clonal structural rearrangements of chromosomes 1, 2, 11, 15, 18, 19 and of chromosomes 10, 20 were also found in LRec-1 sf and LRec-1 cells, accordingly. There were no new clonal structural chromosome rearrangements in LRec-1 k and LRec-3 cells. We compared locies of chromosomes involved in rearrangements with mapped genes on these chromosomes according to RATMAP. Supposedly these genes are involved in spontaneous immortalization of rat embryo fibroblast and malignant transformation of LRec-1 and LRec-3 cells and rearrangements of chromosomes 1, 2, 11, 15 and 18 facilitate expression of growth factors of LRec-1 sf cells.     

The gene and the pseudogene for mouse p53 cellular tumor antigen are located on different chromosomes.

The chromosomal assignments of the two genes encoding the murine p53 cellular tumor antigen were determined by using a panel of mouse-Chinese hamster somatic cell hybrid clones and a mouse p53-specific cDNA clone. One gene, probably the functional member of the family, was found to be on chromosome 11. The other gene, which is probably a processed pseudogene, was assigned to chromosome 14. The potential relevance of these findings to documented cases of chromosome 11 trisomy are also discussed.     

Differentiation stage-specific analysis of gene function with inducible short hair-pin RNA in differentiating embryonic stem cells

Cell differentiation is regulated by spatial and temporal coordination of gene expressions. Previously, we have established an embryonic stem (ES) cell differentiation system that can trace early cardiovascular developmental process in vitro. Here we show that tetracycline-induced short hair-pin RNA (shRNA) expression in differentiating ES cells successfully suppressed stage-specific genes for differentiation and modified cell fates. We established ES cell lines carrying shRNA gene driven by tRNA(val) promoter with tetracycline operator sequences (tet-ON system). When expression of vascular endothelial growth factor receptor-2 (VEGFR2) gene, a vascular progenitor and mesoderm marker and an essential gene for endothelial cell (EC) differentiation, was suppressed by shRNA in early ES cell differentiation, appearance of VEGFR2(+) mesoderm cells was substantially reduced. Suppression of VEGFR2 expression at mesoderm stage almost completely inhibited EC differentiation from VEGFR2(+) mesoderm cells. This novel experimental system, thus, can selectively determine stage-specific roles of genes in differentiation in vitro.     

Generation of reporter hESCs by targeting EGFP at the CD144 locus to facilitate the endothelial differentiation

Reporter embryonic stem cell (ESC) lines with tissue‐specific reporter genes may contribute to optimizing the differentiation conditions in vitro as well as trafficking transplanted cells in vivo. To optimize and monitor endothelial cell (EC) differentiation specifically, here we targeted the enhanced green fluorescent protein (EGFP) reporter gene at the junction of 5′UTR and exon2 of the endothelial specific marker gene CD144 using TALENs in human ESCs (H9) to generate a EGFP‐CD144‐reporter ESC line. The reporter cells expressed EGFP and CD144 increasingly and specifically without unexpected effects during the EC differentiation. The EC differentiation protocol was optimized and applied to EC differentiation from hiPSCs, resulting in an efficient and simplified endothelial differentiation approach. Here we created our own optimized and robust protocol for EC differentiation of hESCs and hiPSCs by generating the lineage‐specific site‐specific integration reporter cell lines, showing great potential to be applied in the fields such as trafficking gene and cell fate in vivo in preclinical animal models.     

Complete or partial trisomy for the long arm of chromosome 1 in patients with various hematologic malignancies

This study contains data obtained from a cytogenetic investigation of six patients with acute and chronic leukaemia. The karyotypes of bone marrow or blood cells of these patients showed a partial or complete trisomy for the long arm of chromosome 1. Three observations revealed a pronounced resistance of cell clones with 1q+ towards cytostatic therapy, and a comparatively short life span of patients after detection of 1q+. The importance of these changes for the role of some chromosomes and chromosome loci in leukaemogenesis is discussed.     

Gene expression variation increase in trisomy 21 tissues

Congenital development disorders with variable severity occur in trisomy 21. However, how these phenotypic abnormalities develop with variations remains elusive. We hypothesize that the differences in euploid gene expression variation among trisomy 21 tissues are caused by the presence of an extra copy of chromosome 21 and may contribute to the phenotypic variations in Down syndrome. We used DNA microarray to measure the differences in gene expression variance between four human trisomy 21 and six euploid amniocytes. The three publicly available data sets of fetal brains, adult brains, and fetal hearts were also analyzed. The numbers of euploid genes with greater variance were significantly higher in all four kinds of trisomy 21 tissues (p < 0.01) than in the corresponding euploid tissues. Seventeen euploid genes with significantly different variance between trisomy 21 and euploid amniocytes were found using the F test. In summary, there is a set of euploid genes that shows greater variance of expression in human trisomy 21 tissues than in euploid tissues. This change may contribute to producing the variable phenotypic abnormalities observed in Down syndrome.     

In vitro culture conditions to study keratinocyte differentiation using the HaCaT cell line

In vitro models to study the process of keratinocyte differentiation have been hindered by the stringent culture requirements and limitations imposed by the inherent properties of the cells. Primary keratinocytes only have a finite life span, while transformed cell lines exhibit many phenotypic features not found in normal cells. The spontaneously immortalized HaCaT cell line has been a widely employed keratinocyte model due to its ease of propagation and near normal phenotype, but protocols for differentiation and gene delivery into HaCaT cells vary widely in the literature. Here we report culture conditions for maintaining HaCaT cells in a basal-like state, for efficient differentiation of these cells, and for delivery of transgenes by transfection or adenoviral infection. This technological report will provide guidance to a large audience of scientists interested in investigating mechanisms of differentiation and skin morphogenesis.     

Phenotypic diversity in cultured cerebral microvascular endothelial cells

Summary Diversity exists in both the structure and function of the endothelial cells (EC) that comprise the microvasculature of different organs. Studies of EC have been aided by our ability to first isolate and subsequently establish cultures from microvascularized tissue. After the isolation of microvessel endothelial cells (MEC) derived from rat cerebrum, we observed morphologic differences in colonies of cells that grew in primary cultures. The morphologies ranged from a cobblestone phenotype considered typical of EC in culture to elongated and stellate cell appearances. Serially passaged cell lines were established based on two parameters: initially by growth and, second, on differences in primary colony morphology using selective weeding techniques. Each culture was examined for the presence of EC-characteristic markers which include Factor-VIII-related antigen, angiotensin-I-converting enzyme activity, collagen type IV synthesis, and PGI₂ production. Variable expression of each of these characteristics among the established EC lines was observed. Growth curves established for each of the EC cultures demonstrated differences in both population doubling rates and cell densities at confluence. The endocytic capacity of each EC line was also evaluated. Our ability to isolate and establish a number of morphologically distinct EC cultures indicates that diversity exists within the EC that comprise the cerebral microvasculature. Diversity in the established cell lines suggests either the EC that line the brain microvasculature exist as a mosaic or that morphologically distinct cultures may originate from different microanatomical origins (arteriolar, true capillary, or venular) or may have resulted from cells at different points in their in vitro life spans at the time of isolation. This research was supported by grants HLO3227 and HLO1514 from the National Institutes of Health, Bethesda, MD.     

Immortalization of bovine umbilical vein endothelial cells: a model for the study of vascular endothelium

Endothelial cells perform a large array of physiological functions that are influenced by their cellular heterogeneity in the different vascular beds. Vein endothelial cells isolated from the umbilical cords are commonly used to study vascular endothelium. Primary cultures of these cells, however, have low proliferative capacity and a limited life span. We have immortalized bovine umbilical vein endothelial cells (BUVEC) by transfection with an expression vector containing the human papillomavirus type 16 E6E7 oncogenes. Expression of E6E7 extended the life span of BUVEC from 40 to more than 1-20 cell replication cycles with no signs of senescence. Four immortalized clones were isolated and found to maintain endothelial cell properties, such as the uptake of acetylated low density lipoprotein, the expression of the von Willebrand protein, the binding of endothelial cell-specific lectins and proliferative responses to the specific endothelial cell mitogen, vascular endothelial growth factor. Moreover, clone BVE-E6E7-1, like its wild-type counterparts, expressed prolactin mRNA and decreased its proliferation in response to the anti-angiogenic 16-kDa fragment of prolactin. This clone showed little signs of genetic instability as revealed by centrosome and chromosome number analysis. Thus, immortalized E6E7 BUVEC cell lines retain endothelial cell characteristics and could facilitate studies to investigate the action of regulatory factors of vascular endothelium. Moreover, being the first non-human umbilical vein endothelial cell lines, their use should provide insights into the mechanisms governing species-related heterogeneity of endothelial cells.     

Isolation and mapping of 62 new RFLP markers on human chromosome 11.

To obtain new RFLP markers on human chromosome 11 for a high-resolution map, we constructed a cosmid library from a Chinese hamster x human somatic hybrid cell line that retains only human chromosome 11 in a Chinese hamster genomic background. A total of 3,500 cosmids were isolated by colony hybridization with labeled human genomic DNA. DNA was prepared from 130 of these cosmid clones and examined for RFLP. In 62 of them, polymorphism was detected with one or more enzymes; four RFLPs were VNTR systems. All polymorphic clones were assigned to one of 22 intervals obtained by mapping on a deletion panel of 15 somatic hybrid cell lines containing parts of chromosome 11; 11 clones were finely mapped by in situ hybridization. Although RFLP markers were scattered on the whole chromosome, they were found predominantly in the regions of R-banding. These DNA markers will contribute to fine mapping of genes causing inherited disorders and tumor-suppressor genes that reside on chromosome 11. Furthermore, as one-third of the cosmid clones revealed a band or bands in Chinese hamster DNA, indicating sequence conservation, this subset of clones may be useful for isolating biologically important genes on chromosome 11.     

Assignment of human uroporphyrinogen I synthase locus to region 11qter by gene dosage effect

Summary Recently Meisler et al. (1980) reported the results of mouse/human somatic cell hybrid studies which indicated that the locus for human uroporphyrinogen I synthase (UPS) (EC 4.3.1.8) maps to chromosome 11. To evaluate further this assignment we have studied the expression of this enzyme in red cells of three children with a trisomy of the region 11qter. We confirm the results of Meisler et al. (1980) and demonstrate that uroporphyrinogen I synthase activity is increased by a factor of 1.5 in trisomy 11qter. In erythrocytes of one child with a trisomy 11p, the expression of this enzyme was normal.