Phenotypic characterization of telomerase-immortalized primary non-malignant and malignant tumor-derived human prostate epithelial cell lines

In vitro human prostate cell culture models are critical for clarifying the mechanism of prostate cancer progression and for testing preventive and therapeutic agents. Cell lines ideal for the study of human primary prostate tumors would be those derived from spontaneously immortalized tumor cells; unfortunately, explanted primary prostate cells survive only short-term in culture, and rarely immortalize spontaneously. Therefore, we recently have generated five immortal human prostate epithelial cell cultures derived from both the benign and malignant tissues of prostate cancer patients with telomerase, a gene that prevents cellular senescence. Examination of these cell lines for their morphologies and proliferative capacities, their abilities to grow in low serum, to respond to androgen stimulation, to grow above the agar layer, to form tumors in SCID mice, suggests that they may serve as valid, useful tools for the elucidation of early events in prostate tumorigenesis. Furthermore, the chromosome alterations observed in these immortalized cell lines expressing aspects of the malignant phenotypes imply that these cell lines accurately recapitulate the genetic composition of primary tumors. These novel in vitro models may offer unique models for the study of prostate carcinogenesis and also provide the means for testing both chemopreventive and chemotherapeutic agents.     

In situ amplification of DNA fragments specific for human Y chromosome in cellular nuclei by PCR

Using single primer pairs Y3 and Y4, in siru polymerase chain reaction (in situ PCR) was successfully performed on the specimen slides of peripheral leukocytes. By both of the direct digpxiginin-11-dUTP incorporation into PCR products with in situ PCR (direct in situ PCR) and in situ PCR followed by detection of in situ hybridization (indirect in siru PCR), DNA fragments specific for human Y chromosome were obviously amplified in cellular nuclei of specimens on the slides. The results were verified by Southern analysis. The methodology of in situ PCR and its application were discussed.     

Characterization of cancer cell lines established from two human metastatic breast cancers

Summary Cell lines are valuable resources for the study of the malignancy and potential therapy of human breast cancer. A major problem with adapting fresh breast tumor specimens to grow in vitro is contamination by fibroblasts. Previously, we have reported a technique to overcome this problem (Nayak, S. K; Dillman, R. O. Clin. Biotechnol. 3:237–242; 1991). We have recently established two new breast cancer cell lines, HH315 and HH375, that were derived from abdominal and supraclavicular lymph node metastases from two patients. They were characterized by (1) growth kinetics; (2) staining with monoclonal antibodies (MoAbs) to cytokeratin-19, epithelial membrane antigen (EMA), anticarcinoembryonic antigen (CEA), breast cancer antigen 1 (BRST-1), breast cancer antigen 2 (BRST-2), Her2/neu, and p53; (3) expression of domains of urinary plasminogen activator (uPA), neural cell adhesion molecule (NCAM), and haptoglobin (Hp) (Harvey et al., 1997); and (4) karyotypic analysis. Growth kinetic studies showed that doubling times for both lines ranged from 48 to 96 h. These two cell lines were found to have characteristics of the metastatic breast cancer cells. Both lines stained positive with MoAbs to cytokeratin-19 and EMA, thus confirming their epithelial origin. They also strongly reacted with the pan-breast carcinoma MoAbs BRST-1 and BRST-2, and carcinoembryonic CEA MoAb. Both cell lines overexpressed the oncogene proteins Her2/neu and p53. The tumor cells were negative for estrogen and progesterone receptors. HH315 cells were poorly differentiated, whereas the HH375 cells exhibited adenocarcinoma morphology. Both cell lines showed intense cell surface and some cytoplasmic staining for uPA, NCAM, and Hp domains, which is a characteristic of malignant neoplasms (Harvey et al., 1997). The HH375 cell line showed two cell types, of which 60% were hyperdiploids with 60–70 chromosomes and 5–10 marker chromosomes. The remaining cells were polyploid with more than 200 chromosomes. Cell line HH315 consisted of only a polyploid population. These cell lines may be useful in breast cancer research.     

Conservation of human Y chromosome sequences among male great apes: Implications for the evolution of Y chromosomes

Nine newly described single-copy and lowcopy-number genomic DNA sequences isolated from a flow-sorted human Y chromosome library were mapped to regions of the human Y chromosome and were hybridized to Southern blots of male and female great ape genomic DNAs (Gorilla gorilla, Pan troglodytes, Pongo pygmaeus). Eight of the nine sequences mapped to the euchromatic Y long arm (Yq) in humans, and the ninth mapped to the short arm or pericentromeric region. All nine of the newly identified sequences and two additional human Yq sequences hybridized to restriction fragments in male but not female genomic DNA from the great apes, indicating Y chromosome localization. Seven of these 11 human Yq sequences hybridized to similarly-sized restriction endonuclease fragments in all the great ape species analyzed. The five human sequences that mapped to the most distal subregion of Yq (deletion of which region is associated with spermatogenic failure in humans) were hybridized to Southern blots generated by pulsed-field gel electrophoresis. These sequences define a region of approximately 1 Mb on human Yq in which HpaII tiny fragment (HTF) islands appear to be absent. The conservation of these human Yq sequences on great ape Y chromosomes indicates a greater stability in this region of the Y than has been previously described for most anonymous human Y chromosomal sequences. The stability of these sequences on great ape Y chromosomes seems remarkable given that this region of the Y does not undergo meiotic recombination and the sequences do not appear to encode genes for which positive selection might occur. Correspondence to: B. Steele Allen     

Karyotype variation of CHO host cell lines over time in culture characterized by chromosome counting and chromosome painting

Genomic rearrangements are a common phenomenon in rapidly growing cell lines such as Chinese hamster ovary (CHO) cells, a feature that in the context of production of biologics may lead to cell line and product instability. Few methods exist to assess such genome wide instability. Here, we use the population distribution of chromosome numbers per cell as well as chromosome painting to quantify the karyotypic variation in several CHO host cell lines. CHO‐S, CHO‐K1 8 mM glutamine, and CHO‐K1 cells adapted to grow in media containing no glutamine were analyzed over up to 6 months in culture. All three cell lines were clearly distinguishable by their chromosome number distribution and by the specific chromosome rearrangements that were present in each population. Chromosome Painting revealed a predominant karyotype for each cell line at the start of the experiment, completed by a large number of variants present in each population. Over time in culture, the predominant karyotype changed for CHO‐S and CHO‐K1, with the diversity increasing and new variants appearing, while CHO‐K1 0 mM Gln preferred chromosome pattern increased in percent of the population over time. As control, Chinese hamster lung fibroblasts were shown to also contain an increasing number of variants over time in culture.     

用C-带和涂染技术检测棕色田鼠Y染色体

采用染色体C 带技术和小鼠整条Y染色体特异探针检测棕色田鼠的Y染色体 ,结果如下 :棕色田鼠雄性个体C 带中期分裂相中 ,X性染色体是亚中部着丝粒染色体 ,在着丝粒处存在着强烈的C阳性带 ,而且在短臂的中间也有一条C阳性带 ,但是没有发现深染的Y染色体。用小鼠整条Y染色体特异探针涂染棕色田鼠的骨髓细胞中期分裂相和间期核 ,以小鼠骨髓细胞中期分裂相和间期核作为对照。涂染结果表明 :棕色田鼠骨髓细胞中期分裂相和间期核涂染信号检出率分别为 0 - 2 %和 3% - 5 % ,两者均呈阴性反应 ,而对照都呈阳性反应。根据实验结果 ,作者认为在棕色田鼠的Y染色体上及整个基因组DNA中不存在小鼠整条Y染色体特异DNA的同源序列 ,其Y染色体上可能没有决定雄性性别的重要基因     

Cell fate regulation by reticulon-4 in human prostate cancers

Reticulon-4 (RTN4), a reticulon family protein localized in the endoplasmic reticulum, is reported to be involved in multiple physiological processes like neuroendocrine secretion and membrane trafficking in neuroendocrine cells. Previous studies have presented a great potential of RTN4 for the treatment of autoimmune-mediated demyelinating diseases and spinal cord injury regeneration. While interaction with Bcl-2 and Bcl-2-like family in apoptosis modulation implicated its possible role in various human cancers. However, the investigation of this gene in prostate cancer is mainly ignored. Here in our current study, we focused on its role in prostate cancer and found that RTN4 DNA copy numbers were higher in prostate cancer than normal prostate gland while its RNA and protein expressions were relatively lower. Chromosomal neighbor gene EML6 had similar expression patterns with RTN4 in prostate cancer tissues and cell lines, and further research found that they could be both targeted by miR-148a-3p. Lentivirus-mediated RTN4 overexpression potently inhibited DU145 and LNCaP cells proliferation. Cell cycle was blocked in G2/M phase and significant cell senescence was observed in RTN4 overexpressed prostate cancer cells. Finally, interaction networks in the normal prostate gland and cancer tissues further revealed that RTN4 maybe phosphorylated by MAPKAPK2 and FYN at tyrosine 591 and serine 107, respectively. All these results implied that RTN4 might somehow participate in prostate tumor progression, and this elicits possibility to develop or identify selective agents targeting RTN4 for prostate cancer therapy.     

Loss of chromosome Y in primary tumors

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Synergistic chromatin repression of the tumor suppressor gene RARB in human prostate cancers

DNA methylation and polycomb proteins are well-known mediators of epigenetic silencing in mammalian cells. Usually described as mutually exclusive, this statement is today controversial and recent in vitro studies suggest the co-existence of both repressor systems. We addressed this issue in the study of Retinoic Acid Receptor β (RARβ), a tumor suppressor gene frequently silenced in prostate cancer. We found that the RARβ promoter is hypermethylated in all studied prostate tumors and methylation levels are positively correlated with H3K27me3 enrichments. Thus, by using bisulfite conversion and pyrosequencing of immunoprecipitated H3K27me3 chromatin, we demonstrated that DNA methylation and polycomb repression co-exist in vivo at this locus. We found this repressive association in 6/6 patient tumor samples of different Gleason score, suggesting a strong interplay of DNA methylation and EZH2 to silence RARβ during prostate tumorigenesis.     

Secretome derived from breast tumor cell lines alters the morphology of human umbilical vein endothelial cells

Metastases, responsible for most of the solid tumor associated deaths, require angiogenesis and changes in endothelial cells. In this work, the effect of the secretomes of three breast tumor cell lines (MCF-7, MDA-MB-231 and ZR-75-30) on human umbilical vein endothelial cells (HUVEC) morphology was investigated. HUVEC treated with secretomes from breast cells were analyzed by confocal and time-lapse microscopy. Secretomes from ZR-75-30 and MDA-MB-231 cells modify the morphology and adhesion of HUVEC. These changes may provoke the loss of endothelial monolayer integrity. In consequence, tumor cells could have an increased access to circulation, which would then enhance metastasis.     

Overexpression of the c-erbB-2 protein in human breast tumor cell lines

The c-erbB-2 proto-oncogene is amplified in a high percentage of primary human breast tumors, suggesting that the overexpression of this gene may be involved in the development of human breast cancer. We have investigated five human breast tumor cell lines and have detected amplified c-erbB-2 gene copies in two of them. This amplification leads to overexpression of the c-erbB-2 protein. In addition, two other cell lines have elevated protein levels without gene amplification, suggesting that other mechanisms can lead to overexpression of the c-erbB-2 protein. These results are similar to those that we obtained during a study of primary breast tumors (Berger et al.: Cancer Res 48:1238-1243, 1988). These breast tumor cell lines should be useful for an analysis of c-erbB-2 expression and of the mechanisms that in some cases lead to overexpression.     

Epigenetic inactivation of PCDH10 in human prostate cancer cell lines

PCDH10 (protocadherin-10), a novel tumour suppressor gene, is down-regulated in several human cancers due to hypermethylation of promoter CGIs (CpG islands). Here, we investigated the expression of PCDH10 in different normal adult tissues and in a panel of prostate cancer cell lines. PCDH10 was widely expressed in normal tissues with higher levels in the prostate. The expression of PCDH10 was markedly reduced or silenced in prostate cancer cell lines compared with normal adult prostate tissue. Decreased PCDH10 expression was correlated with the methylation status of the PCDH10 promoter. Furthermore, the DNA demethylating agent 5'-azacytidin restored PCDH10 expression by suppressing PCDH10 promoter methylation in prostate cancer cell lines. Treatment with Trichostatin A alone had no significant effect on the expression of PCDH10 but enhanced the effect of 5'-azacytidin. In conclusion, we found that the decreased PCDH10 expression in prostate cancer cells was associated with the aberrant methylation of PCDH10 promoter CGI. Our results may contribute to the understanding of the role of PCDH10 inactivation in the progression of prostate cancers.     

CYP24 splicing variants are associated with different patterns of constitutive and calcitriol-inducible CYP24 activity in human prostate cancer cell lines

24-Hydroxylase (CYP24) activity modulates in vitro and in vivo calcitriol metabolism and biologic effects. We have investigated, in human PC3, DU145 and LNCaP prostate cancer cell lines, the relationship of CYP24 single nucleotide polymorphisms (SNPs) and splicing and the variable patterns of baseline and calcitriol-inducible CYP24 activity. DU145 cells exhibit baseline CYP24 activity that is further induced by calcitriol. Baseline and inducible CYP24 activity were barely detectable in LNCaP cells. In PC3, baseline CYP24 activity was undetectable but induced by calcitriol. A different pattern of SNPs was identified at positions 24, 46, 146 and 198 in the intron between exons 9 and 10 of CYP24 gene in each cancer cell line. DU145 displayed baseline CYP24 splicing between exon 9 and exon 11; splicing was only observed in calcitriol treated LNCaP cells. Untreated PC3 had a mixed picture (splicing and no splicing); only the spliced form was seen after calcitriol treatment. These results demonstrate that calcitriol treatment modulates CYP24 splicing, and suggests that differences in CYP24 splicing are associated with different patterns of CYP24 activity.     

Growth of human tumor cell lines in transferrin-free,low-iron medium

Summary Iron is essential for tumor cell growth. Previous studies have demonstrated that apart from transferrin-bound iron uptake, mammalian cells also possess a transport system capable of efficiently obtaining iron from small molecular weight iron chelates (Sturrock et al., 1990). In the present study, we have examined the ability of tumor cells to grow in the presence of low molecular weight iron chelates of citrate. In chemically defined serum-free medium, most human tumor cell lines required either transferrin (5 μg/ml) or a higher concentration of ferric citrate (500 μM) as an iron source. However, we have also found that from 13 human cell lines tested, 4 were capable of long-term growth in transferrin-free medium with a substantially lower concentration of ferric citrate (5 μM). When grown in medium containing transferrin, both regular and low-iron dependent cell lines use transferrin-bound iron. Growth of both cell types in transferrin medium was inhibited to a certain degree by monoclonal antibody 42/6, which specifically blocks the binding of transferrin to the transferrin receptor. On the contrary, growth of low-iron dependent cell lines in transferrin-free, low-iron medium (5 μM ferric citrate) could not be inhibited by monoclonal antibody 42/6. Furthermore, no autocrine production of transferrin was observed. Low-iron dependent cell lines still remain sensitive to iron depletion as the iron(III) chelator, desferrioxamine, inhibited their growth. We conclude that low-iron dependent tumor cells in transferrin-free, low-iron medium may employ a previously unknown mechanism for uptake of non-transferrin-bound iron that allows them to efficiently use low concentrations of ferric citrate as an iron source. The results are discussed in the context of alternative iron uptake mechanisms to the well-characterized receptor-mediated endocytosis process.     

Y chromosome conserved anchored tagged sequences (YCATS) for the analysis of mammalian male-specific DNA

Y chromosome haplotyping based on microsatellites or single nucleotide polymorphisms has recently proven to be a powerful approach for evolutionary studies of human populations, and also holds great promise for the studies of wild species. However, the use of the approach is hampered in most natural populations by the lack of Y chromosome markers and sequence information. Here, we report the large-scale development of Y chromosome conserved anchor tagged sequence (YCATS) markers in mammals by a polymerase chain reaction screening approach. Exonic primers flanking 48 different introns of Y-linked genes were developed based on human and mouse sequences, and screened on a set of 20 different mammals. On average about 10 introns were amplified for each species and a total of 100 kb of Y chromosome sequence were obtained. Intron size in humans was a reasonable predictor of intron size in other mammals (r2 = 0.45) and there was a negative correlation between human fragment size and amplification success. We discuss a number of factors affecting the possibility of developing conserved Y chromosome markers, including fast evolution of Y chromosome sequences due to male-biased mutation and adaptive evolution of male-specific genes, dynamic evolution of the Y chromosome due to being a nonrecombining unit, and homology with X chromosome sequences.     

Synergistic chromatin repression of the tumor suppressor gene RARB in human prostate cancers

DNA methylation and polycomb proteins are well-known mediators of epigenetic silencing in mammalian cells. Usually described as mutually exclusive, this statement is today controversial and recent in vitro studies suggest the co-existence of both repressor systems. We addressed this issue in the study of Retinoic Acid Receptor β (RARβ), a tumor suppressor gene frequently silenced in prostate cancer. We found that the RARβ promoter is hypermethylated in all studied prostate tumors and methylation levels are positively correlated with H3K27me3 enrichments. Thus, by using bisulfite conversion and pyrosequencing of immunoprecipitated H3K27me3 chromatin, we demonstrated that DNA methylation and polycomb repression co-exist in vivo at this locus. We found this repressive association in 6/6 patient tumor samples of different Gleason score, suggesting a strong interplay of DNA methylation and EZH2 to silence RARβ during prostate tumorigenesis.     

Y chromosome microsatellite markers identified from the tammar wallaby (Macropus eugenii) and their amplification in three other macropod species

Microsatellites were identified from three fully sequenced Y chromosome‐specific bacterial artificial chromosome clones from the tammar wallaby, Macropus eugenii. Ten microsatellites were genotyped in male tammar wallabies. Four loci were polymorphic with between two and six alleles per locus. Eleven different haplotypes were identified from 22 male tammar wallabies. No amplifications were obtained from female samples. Each microsatellite was also shown to amplify reliably in at least one other macropod species. These markers may therefore prove useful as some of the first male‐specific genetic markers for marsupials, with potential application to studies of male‐biased dispersal and mating systems.     

The proliferation of human tumor cell lines in the presence of different agars,agaroses, and methyl cellulose

Summary Human tumor cell lines, derived from cancers of the colon, ovary, and cervix, were grown in liquid tissue culture media and media made semisolid with agar (Bacto & deoxycholate lactose agar), agarose [LE, ME, Sea Plaque and Sea Prep (15/45)], and methyl cellulose. The effects of each agent on overall cell proliferation and rate of overall cell proliferation were examined. The agents, used to make media semisolid, were observed to inhibit or, in some cases, enhance cell growth in a fashion that was characteristic of individual cell lines. These phenomena may be of consequence to the optimization of nutrient media for primary tumor cell preparations. This work was supported by the Veteran's Administration.     

Development and characterization of new immortalized human breast cell lines

New human breast cell lines were developed from metastatic breast cancer tissues and normal breast tissues. Primary cultures were initiated from cellular outgrowths of explanted tissues or from mechanically isolated cells in two serum-free media. Cell cultures derived from both cancer and normal tissues were immortalized with pRSV-T plasmid to generate permanent breast cell lines that exhibited an epithelial morphology. Cell lines generated in this study were characterized with respect to morphology, growth rate, karyotype, presence of specific genes, and the expression of epithelial and breast markers. The cell lines expressed the epithelial cell markers, cytokeratins 8 and 18, and retained the capacity to produce human milk fat globulin. They also express the BRCA-1, erbB2, and EGF receptor genes and possess the H-ras, K-ras, and p53 genes. Preliminary data showed that one of the new cancer cell lines was highly sensitive to the cytotoxic action of taxol. It is envisioned that the new breast cell lines will be useful as targets for identification of therapeutic agents against breast cancer and as models for carcinogenesis studies. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cellular localization of tissue factor in human breast cancer cell lines

Expression of tissue factor (TF), the cellular receptor of clotting factor VII/VIIa, is a feature of certain malignant tumours. The TF gene has been classified as an immediate early gene responsive to serum and cytokines. Thus, the regulation of TF gene expression seems to play a role in cell growth. Recently, we have shown that constitutive TF expression in MCF-7 breast cancer cells is modulated by such growth factors as EGF, TGFα, and IL-1. The present study deals with the immunocytochemically detectable cellular distribution of TF in human breast cancer cell lines MCF-7 and MaTu stimulated by EGF and TGFα. In MCF-7 cells growing logarithmically, stimulation led to a significant increase of TF mRNA after 2 h (in situ hybridization, Northern blot) and to maximum TF expression after 6 h (immunohistochemistry). When decorated by monoclonal antibodies, TF protein showed a pronounced localization at ruffled membrane areas, cell edges, and processes of spreading cells after 6 and 20 h. In more flattened cells TF was concentrated in peripheric lamellae and microspikes communicating with neighbouring cells. After epithelial colony pattern had established, TF was predominantly accumulated at the intercellular boundaries. The vary same distribution patterns as seen in MCF-7 cells were true for the stimulated MaTu cell line. The dynamics and cellular distribution patterns of stimulated TF expression support the hypothesis that TF could be of importance for morphogenic events associated with the growth and differentiation of breast cancer cells in culture.