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Incidence of colon cancer has increased rapidly in China. Although many colon cancer cell lines have been established previously, most of them were derived from patients from western countries. Epidemiological, clinical, cytogenetic, and molecular biological studies showed that there are considerable differences between Chinese and western countries colon cancer patients. Therefore, establishment of novel colon cancer cell line from Chinese is useful for studying the racial difference of this disease and can be important for studying the pathogenesis of colon cancer in China. In our laboratory, two novel continuous human colon cancer cell lines, SHT-1 and SHH-1, have been established in vitro from Chinese patients, and both cell lines have been passaged for 4 yr, and they have been continuously subcultured with more than 800 population doubling and without signs of senescence. Both cell lines were obtained from primary tumor tissues during colon cancer surgery. Cells grew rapidly with a doubling time of 36–39 h and a plating efficiency of 26–28%. These cells exhibited an epithelial morphology and expressed cytokeratin. Tumor developed in severe combined immunodeficient (SCID) mice 4–6 wk after inoculated subcutaneously with the cultured cancer cells. Karyotypic analysis and comparative genomic hybridization (CGH) analysis in SHT-1 cells revealed a hypertriploid modal number of 76 with numerous numerical and structural abnormalities previously linked to colon cancer. In another cell line (SHH-1), CGH analysis revealed that −1p13 was the only cytogenetic anomaly.
Tissue microarrays are ordered arrays of hundreds to thousands of tissue cores in a single paraffin block. We invented a novel method to make a high-throughput micro-array group. Conventional smaller tissue microarrays were made first and then sectioned. Separate paraffin films were arrayed orderly onto a regular-sized glass slide to form a larger microarray group. Sections were not floated in a water bath but, rather, were cut singly using conventional microtome, arrayed orderly onto the glass slide with forceps instead of using a tape-based tissue transfer system, and then unfolded with warm water (46° C) using a micropipette. This not only lowers the difficulty in sectioning but the overall tissue disks can be included in the same section. A micro-array group of 2,534 small disks (theoretically, 2,560 disks can be made; 26 fell off during the procedure), the most up to now, was successfully made and may be used in immuno-histochemistry, mRNA in situ hybridization, and flourescent in situ hybridization.
Mycoplasma contamination affects many different aspects of cell culturing, resulting in unreliable experimental results and potentially harmful biological products. Therefore, the specificity, sensitivity, and reliability of detecting mycoplasma contamination are important aspects of quality control in biotechnological products. In this study, Mycoplasma hyorhinis was adopted as a model strain to evaluate the effects of storage on the viability of Mycoplasma species in cell culture samples. Medium X was compared with conventional media 243 and 988 for the ability to detect M. hyorhinis. The 101 CFU/ml of M. hyorhinis was inoculated into medium X prepared using the same lots of components and preserved for 7 d, 1 mo, and 2 mo. M. hyorhinis grew readily and typically on agar plates prepared within 1 mo. The viable mycoplasmas in samples containing different initial titers (101 and 106 CFU/ml) after storage at 4° C and −30° C were analyzed. During storage, viable organisms were found with little or no reduction in titers after storage for 8 wk at −30° C under aerobic and anaerobic conditions. A reduction in titers of 3 log10 occurred after 4 wk storage for high-dose cultures (106 CFU/ml) at 4° C. The titers of viable organisms were diminished over 8 wk at 4° C under aerobic and anaerobic conditions.
We have recently reported the induction of dental pulp stem cells (DPSCs) into dentin-secreting odontoblast-like cells after stimulation by isolated dentin matrix components, thus mimicking the nature of tissue regeneration seen after tooth disease and injury. After confluency, the cells were further cultured for 21 d in the 10% fetal bovine serum (FBS) Dulbecco's modified Eagle's medium (DMEM) (control), and in this medium, with the addition of dentin extract (DE) and the mineralization supplement (MS) of ascorbic acid and β-glycerophosphate (treatment). To identify genes associated with this process, specimens were analyzed with a HG-U133A human gene chip and Arrayassist software. A total of 425 genes, among them 21 matrix and eight TGF-β-related genes, were either up- or downregulated in the experimental group in which the cells showed odontoblast-like differentiation and mineralization. Expression of selected genes was further confirmed by real-time polymerase chain reaction (PCR) analysis. Of the extracellular matrix (ECM)-related genes, two types of collagen genes were upregulated and seven others down-regulated. Other ECM-related genes, for example fibulin-1, tenascin C, and particularly thrombospondin 1, were upregulated, and fibulin-2 was downregulated. Most noticeably, the matrix metalloproteinase 1 was induced by the treatment. In the TGF-β superfamily, upregulation of the type II receptor, endoglin, and growth/differentiation factor 5 was coordinated with the downregulation of activin A, TGF-β2, and TGF-β1 itself. This study identifies the matrix and TGF-β-related gene profiles during the DPSC cell mineralization in which several genes are reported for the first time to be associated with this process, thus greatly expanding our molecular knowledge of the induced disease repair process.
Human mesenchymal stem cells (hMSCs) are expected to be an enormous potential source for future cell therapy, because of their self-renewing divisions and also because of their multiple-lineage differentiation. The finite lifespan of these cells, however, is a hurdle for clinical application. Recently, several hMSC lines have been established by immortalized human telomerase reverse transcriptase gene (hTERT) alone or with hTERT in combination with human papillomavirus type 16 E6/E7 genes (E6/E7) and human proto-oncogene, Bmi-1, but have not so much been characterized their karyotypic stability in detail during extended lifespan under in vitro conditions. In this report, the cells immortalized with the hTERT gene alone exhibited little change in karyotype, whereas the cells immortalized with E6/E7 plus hTERT genes or Bmi-1, E6 plus hTERT genes were unstable regarding chromosome numbers, which altered markedly during prolonged culture. Interestingly, one unique chromosomal alteration was the preferential loss of chromosome 13 in three cell lines, observed by fluorescence in situ hybridization (FISH) and comparative-genomic hybridization (CGH) analysis. The four cell lines all maintained the ability to differentiate into both osteogenic and adipogenic lineages, and two cell lines underwent neuroblastic differentiation. Thus, our results were able to provide a step forward toward fulfilling the need for a sufficient number of cells for new therapeutic applications, and substantiate that these cell lines are a useful model for understanding the mechanisms of chromosomal instability and differentiation of hMSCs.
Mitochondria are the bioenergetic and metabolic centers in eukaryotic cells and play a central role in apoptosis. Mitochondrial distribution is controlled by the microtubular cytoskeleton. The perinuclear aggregation of mitochondria is one of the characteristics associated with some types of cell death. Control of mitochondrial aggregation particularly related to cell death events is poorly understood. Previously, we identified ubiquitously expressed transcript (UXT) as a potential component of mitochondrial associated LRPPRC, a multidomain organizer that potentially integrates mitochondria and the microtubular cytoskeleton with chromosome remodeling. Here we show that when overexpressed in mammalian cells, green fluorescent protein-tagged UXT (GFP-UXT) exhibits four types of distribution patterns that are proportional to the protein level, and increase with time. UXT initially was dispersed in the extranuclear cytosol, then appeared in punctate cytosolic dots, then an intense perinuclear aggregation that eventually invaded and disrupted the nucleus. The punctate cytosolic aggregates of GFP-UXT coincided with aggregates of mitochondria and LRPPRC. We conclude that increasing concentrations of UXT contributes to progressive aggregation of mitochondria and cell death potentially through association of UXT with LRPPRC.
Hepatocyte growth factor (HGF) can induce proliferation and migration of intestinal epithelial cells and has also been shown to be important in wound healing of inflamed mucosal tissues. HGF is known to be expressed along with interleukin-1 (IL-1) by inflamed mucosal tissues, yet the effect of HGF on IL-1-induced proinflammatory cytokine responses by colonic epithelial cells is unknown. In this report, we have examined the effect of HGF on IL-1-induced secretion of IL-8 by the Caco-2 colonic epithelial cell line. HGF stimulation alone had no effect on the secretion of IL-8 by the Caco-2 cells. However, culture of the cells with HGF and suboptimal levels of IL-1 resulted in a significant enhancement of IL-8 secretion compared to cells cultured with IL-1 alone. A similar effect was seen with HGF and IL-1 simulation of monocyte chemoattractant protein-1 secretion by the rat IEC-6 intestinal epithelial cell line. The enhancing effect of HGF was seen regardless of whether the culture medium contained serum or not. Simultaneous stimulation with HGF and IL-1 was required for the enhancing effect as cells pretreated with HGF for 24 h and then stimulated with IL-1 alone secreted IL-8 levels similar to that of cells stimulated with IL-1 alone. These results suggest that in addition to wound healing, HGF may play a role in the IL-1-induced chemokine response of epithelial cells in inflamed mucosal tissues.