Persistence of collagen type II synthesis and secretion in rapidly proliferating human articular chondrocytes in vitro.

Articular chondrocytes (AC) expanded in vitro for tissue engineering rapidly turn off collagen type II (COL2) synthesis. We wanted to inhibit this process sufficiently to obtain therapeutically useful numbers of AC without losing COL2 synthesis. To this end, AC were expanded on their own extracellular matrix (ECM) in structures designated chondrocytes in autologous ECM (CA-ECM). Here, AC maintained a rounded shape and proliferated rapidly. After 13-15 days in culture, 40 x 10(6) cells (median) could be obtained from a cartilage biopsy. Real-time RT-PCR showed a reduced, but persistent, production of COL2A1 mRNA at this time. Flow cytometry showed high levels of intracellular COL2, and immunogold electron microscopy showed high density of well-organized COL2 fibrils in newly synthesized ECM. Interestingly, high levels of COL1A1 mRNA and intracellular protein were detected, but no COL1 was found in the ECM. The slow loss of COL2A1 mRNA was paralleled by a loss of the COL2 regulating transcription factor SOX9 mRNA. Chromatin immunoprecipitation assays could not identify epigenetic histone modifications that would explain the observed changes in COL2 synthesis. Thus, the CA-ECM strategy allows AC to proliferate to clinically useful numbers while maintaining COL2 synthesis and secretion. This strategy may improve tissue engineering of joint surfaces.

Increased expression of visfatin in macrophages of human unstable carotid and coronary atherosclerosis: possible role in inflammation and plaque destabilization.

BACKGROUND: Although the participation of inflammation in atherogenesis is widely recognized, the identification of the different components has not been clarified. In particular, the role of inflammation in plaque destabilization is not fully understood. METHODS AND RESULTS: Our main findings were as follows: (1) In a microarray experiment, we identified visfatin, one of the most recently identified adipokines, as a gene that was markedly enhanced in carotid plaques from symptomatic compared with plaques from asymptomatic individuals. This finding was confirmed when carotid plaques from 7 patients with asymptomatic and 14 patients with symptomatic lesions were examined with real-time reverse transcription polymerase chain reaction. (2) Immunohistochemistry showed that visfatin was localized in areas that were rich in lipid-loaded macrophages. (3) The relationship between visfatin and unstable lesions was also found in patients with coronary artery disease, demonstrating a strong visfatin immunostaining in lipid-rich regions within the material obtained at the site of plaque rupture in patients with acute myocardial infarction. (4) Both oxidized low-density lipoprotein and tumor necrosis factor-alpha increased visfatin expression in THP-1 monocytes, with a particularly enhancing effect when these stimuli were combined. (5) Visfatin increased matrix metalloproteinase-9 activity in THP-1 monocytes and tumor necrosis factor-alpha and interleukin-8 levels in peripheral blood mononuclear cells. Both of these effects were abolished when insulin receptor signaling was blocked. CONCLUSIONS: Our findings suggest that visfatin should be regarded as an inflammatory mediator, localized to foam cell macrophages within unstable atherosclerotic lesions, that potentially plays a role in plaque destabilization.

Isolation and transcription profiling of purified uncultured human stromal stem cells: alteration of gene expression after in vitro cell culture.

Stromal stem cells proliferate in vitro and may be differentiated along several lineages. Freshly isolated, these cells have been too few or insufficiently pure to be thoroughly characterized. Here, we have isolated two populations of CD45-CD34+CD105+ cells from human adipose tissue which could be separated based on expression of CD31. Compared with CD31+ cells, CD31- cells overexpressed transcripts associated with cell cycle quiescence and stemness, and transcripts involved in the biology of cartilage, bone, fat, muscle, and neural tissues. In contrast, CD31+ cells overexpressed transcripts associated with endothelium and the major histocompatibility complex class II complex. Clones of CD31- cells could be expanded in vitro and differentiated into cells with characteristics of bone, fat, and neural-like tissue. On culture, transcripts associated with cell cycle quiescence, stemness, certain cytokines and organ specific genes were down-regulated, whereas transcripts associated with signal transduction, cell adhesion, and cytoskeletal +CD105+CD31- cells from human adipose tissue have stromal stem cell properties which may make them useful for tissue engineering.

Molecular characterizations of derivatives of HCT116 colorectal cancer cells that are resistant to the chemotherapeutic agent 5-fluorouracil.

5-Fluorouracil (5-FU) is the chemotherapeutic drug of choice for the treatment of metastatic colorectal cancer, but resistance to 5-FU remains a major obstacle to successful therapy. We generated 5-FU-resistant derivatives of the HCT116 human colon cancer cell line by serial passage of these cells in the presence of increasing 5-FU concentrations in an attempt to elucidate the biological mechanisms involved in resistance to 5-FU. Two resultant resistant derivatives, HCT116 ResB and ResD, were characterized for resistance phenotypes, genotypes, and gene expression using cells maintained long-term in 5-FU-free media. Compared to parental HCT116 cells that respond to 5-FU challenge by inducing high levels of apoptosis, ResB and ResD derivatives had significantly reduced apoptotic fractions when transiently challenged with 5-FU. ResB and ResD cells were respectively 27- and 121-fold more resistant to 5-FU, had increased doubling times, and significantly increased plating efficiencies compared to the parental cells. Both resistant derivatives retained the wild-type TP53 genotype, TP53 copy number and CGH profile characteristic of the parental line. Alterations in gene expression in the resistant derivatives compared to the parental line were assessed using oligonucleotide microarrays. Overall, the 5-FU-resistant derivatives were characterized by reduced apoptosis and a more aggressive growth phenotype, consistent with the observed up-regulation of apoptosis-inhibitory genes (e.g., IRAK1, MALT1, BIRC5), positive growth-regulatory genes (e.g., CCND3, CCNE2, CCNF, CYR61), and metastasis genes (e.g., LMNB1, F3, TMSNB), and down-regulation of apoptosis-promoting genes (e.g., BNIP3, BNIP3L, FOXO3A) and negative growth-regulatory genes (e.g., AREG, CCNG2, CDKN1A, CDKN1C, GADD45A). 5-FU metabolism-associated genes (e.g., TYMS, DTYMK, UP) and DNA repair genes (e.g., FEN1, FANCG, RAD23B) were also up-regulated in one or both resistant derivatives, suggesting that the resistant derivatives might be able to overcome both 5-FU inhibition of thymidylate synthase and the DNA damage caused by 5-FU, respectively. Development of 5-FU resistance thus appears to encompass deregulation of apoptosis-, proliferation-, DNA repair-, and metastasis-associated regulatory pathways.

Comparison of gene expression in HCT116 treatment derivatives generated by two different 5-fluorouracil exposure protocols.

BACKGROUND: Established colorectal cancer cell lines subjected to different 5-fluorouracil (5-FU) treatment protocols are often used as in vitro model systems for investigations of downstream cellular responses to 5-FU and to generate 5-FU-resistant derivatives for the investigation of biological mechanisms involved in drug resistance. We subjected HCT116 colon cancer cells to two different 5-FU treatment protocols in an attempt to generate resistant derivatives: one that simulated the clinical bolus regimens using clinically-achievable 5-FU levels, the other that utilized serial passage in the presence of increasing 5-FU concentrations (continuous exposure). HCT116 Bolus3, ContinB, and ContinD, corresponding to independently-derived cell lines generated either by bolus exposure or continuous exposure, respectively, were characterized for growth- and apoptosis-associated phenotypes, and gene expression using 8.5 K oligonucleotide microarrays. Comparative gene expression analyses were done in order to determine if transcriptional profiles for the respective treatment derivatives were similar or substantially different, and to identify the signaling and regulatory pathways involved in mediating the downstream response to 5-FU exposure and possibly involved in development of resistance. RESULTS: HCT116 ContinB and ContinD cells were respectively 27-fold and >100-fold more resistant to 5-FU and had reduced apoptotic fractions in response to transient 5-FU challenge compared to the parental cell line, whereas HCT116 Bolus3 cells were not resistant to 5-FU after 3 cycles of bolus 5-FU treatment and had the same apoptotic response to transient 5-FU challenge as the parental cell line. However, gene expression levels and expression level changes for all detected genes in Bolus3 cells were similar to those seen in both the ContinB (strongest correlation) and ContinD derivatives, as demonstrated by correlation and cluster analyses. Regulatory pathways having to do with 5-FU metabolism, apoptosis, and DNA repair were among those that were affected by 5-FU treatment. CONCLUSION: All HCT116 derivative cell lines demonstrated similar transcriptional profiles, despite the facts that they were generated by two different 5-FU exposure protocols and that the bolus exposure derivative had not become resistant to 5-FU. Selection pressures on HCT116 cells as a result of 5-FU challenge are thus similar for both treatment protocols.

Pulmonary and lymph node metastasis is associated with primary tumor interstitial fluid pressure in human melanoma xenografts.

Interstitial fluid pressure (IFP) is elevated in many experimental and human tumors, and high IFP is associated with poor prognosis in human cancer. The significance of elevated IFP in the development of metastatic disease was investigated in the present work by using A-07 human melanoma xenografts as models of cancer in humans. IFP was measured with the micropipette technique (tumor periphery) and the wick-in-needle technique (tumor center). Tumor hypoxia was studied by immunohistochemistry using pimonidazole as a hypoxia marker and by using a radiobiological assay. High central tumor IFP was found to be associated with the development of pulmonary (P = 0.000085) and lymph node (P = 0.000036) metastases in small (150-200 mm(3)) A-07 tumors. Hypoxic cells could not be detected in these tumors. Our study suggests that interstitial hypertension may facilitate tumor cell intravasation and, hence, promote metastasis by mechanisms independent of tumor hypoxia.