Differentiation of cartilage cells studied by quantitative [3H]thymidine autoradiography in vitro

The apical segments of the mandibular condylar cartilage of newborn ICR mice, containing the intact zones of progenitor cells along with a few rows of chondroblasts were initially prelabelled in vitro with [3H]thymidine and were subsequently chased and cultured for as long as eight days. Such explants underwent a process of tissue regeneration, as after three days in culture they reconstituted the original structure of the organ, thus resembling the in vivo appearance of neonatal mandibular condylar cartilage. Cellular proliferation with subsequent differentiation in the regenerating tissue was followed by means of quantitative autoradiography. Immediately after labelling, the autoradiography-positive grains were confined exclusively to progenitor cells. The latter revealed a substantial ability to proliferate in vitro, a fact that was manifested by a progressive increase in the labelling index along the course of the culture. The latter process was followed by cellular differentiation thereby obtaining hypertrophic chondrocytes. The increase in the rate of labelling index and in the total number of [3H]thymidine-labelled cells was significantly correlated with the overall growth of the regenerating explants.     

Normalizing genes for quantitative RT-PCR in differentiating human intestinal epithelial cells and adenocarcinomas of the colon

As for other mRNA measurement methods, quantitative RT-PCR results need to be normalized relative to stably expressed genes. Widely used normalizing genes include beta-actin and glyceraldehyde-3-phosphate dehydrogenase. It has, however, become clear that these and other normalizing genes can display modulated patterns of expression across tissue types and during complex cellular processes such as cell differentiation and cancer progression. Our objective was to set the basis for identifying normalizing genes that displayed stable expression during enterocytic differentiation and between healthy tissue and adenocarcinomas of the human colon. We thus identified novel potential normalizing genes using previously generated cDNA microarray data and examined the alterations of expression of two of these genes as well as seven commonly used normalizing genes during the enterocytic differentiation process and between matched pairs of resection margins and primary carcinomas of the human colon using real-time RT-PCR. We found that ribosomal phosphoprotein P0 was particularly stable in all intestinal epithelial cell extracts, thereby representing a particularly robust housekeeping reference gene for the assessment of gene expression during the human enterocytic differentiation process. On the other hand, beta-2-microglobulin generated the best score as a normalizing gene for comparing human colon primary carcinomas with their corresponding normal mucosa of the resection margin, although others were found to represent acceptable alternatives. In conclusion, we identified and characterized specific normalizing genes that should significantly improve quantitative mRNA studies related to both the differentiation process of the human intestinal epithelium and adenocarcinomas of the human colon. This approach should also be useful to validate normalizing genes in other intestinal contexts.     

Adoptive immunotherapy of a mouse colon carcinoma with recombinant interleukin-2 alone or combined with lymphokine-activated killer cells or tumor-immune lymphocytes

Summary We have used a BALB/c colonic adenocarcinoma (C-26) to evaluate the therapeutic potential of recombinant interleukin-2 (rIL-2) at high and low dosages in combination with or without lymphokine-activated killers (LAK) or tumor-specific, immune lymphocytes in either an adjuvant spontaneous or an artificial metastasis system. Most (80%) of the mice that underwent s.c. C-26 tumor excision were shown to die of spontaneous metastasis with lung involvement by 1–4 months after excision. Postsurgical systemic treatment with low-dose rIL-2 (3 × 10⁴ U/day, i.p.) increased the survival rate to 31% as compared to 21% (not significant) in excised controls while administration of high-dose rIL-2 (8 × 10⁴ U/day) led to 53% survival (P <0.01). Both LAK cells and C-26-tumor-immune lymphocytes given during rIL-2 treatment significantly increased the effects of rIL-2 at the low but not at the high-dose, with tumor-immune effectors resulting in the highest percentage (63%) of cures. When mice bearing 3-day artificial lung metastases of C-26 cells were treated with low- or high-dose rIL-2, in combination with or without LAK or tumor-immune lymphocytes, a highly significant reduction or abrogation of the number of lung foci was observed with all treatments, including those involving or tumor-immune lymphocytes alone. Assessment of survival benefit in these mice, however, showed survival prolongation, with 20% cures achieved by low-dose rIL-2 alone and up to 65% cures by LAK in combination with low-dose rIL-2. In this system of artificial metastasis high-dose rIL-2 alone increased the survival time but failed to cure the animals, and the addition of LAK was ineffective whereas that of tumor-immune lymphocytes led to 80% cure. These results suggest that tumorimmune lymphocytes are more effective than LAK when combined with rIL-2 and that caution is necessary in extrapolating findings obtained in artificial metastasis models.     

Periodic patterns of actin turnover in lamellipodia and lamellae of migrating epithelial cells analyzed by quantitative Fluorescent Speckle Microscopy

We measured actin turnover in lamellipodia and lamellae of migrating cells, using quantitative Fluorescent Speckle Microscopy. Lamellae disassembled at low rates from the front to the back. However, the dominant feature in their turnover was a spatially random pattern of periodic polymerization and depolymerization moving with the retrograde flow. Power spectra contained frequencies between 0.5 and 1 cycle/min. The spectra remained unchanged when applying Latrunculin A and Jasplakinolide in low doses, except that additional frequencies occurred beyond 1 cycle/min. Whereas Latrunculin did not change the rate of mean disassembly, Jasplakinolide halted it completely, indicating that the steady state and the dynamics of actin turnover are differentially affected by pharmacological agents. Lamellipodia assembled in recurring bursts at the leading edge and disassembled approximately 2.5 microm behind. Events of polymerization correlated spatially and temporally with transient formation of Arp2/3 clusters. In lamellae, Arp2/3 accumulation and polymerization correlated only spatially, suggesting an Arp2/3-independent mechanism for filament nucleation. To acquire these data we had to enhance the resolution of quantitative Fluorescent Speckle Microscopy to the submicron level. Several algorithmic advances in speckle image processing are described enabling the analysis of kinetic and kinematic activities of polymer networks at the level of single speckles.     

Expression of lactoperoxidase in differentiated mouse colon epithelial cells

Lactoperoxidase (LPO) is known to be present in secreted fluids, such as milk and saliva. Functionally, LPO teams up with dual oxidases (DUOXs) to generate bactericidal hypothiocyanite in the presence of thiocyanate. DUOX2 is expressed in intestinal epithelium, but there is little information on LPO expression in this tissue. To fill the gap of knowledge, we have analyzed Lpo gene expression and its regulation in mouse intestine. In wild-type (WT) C57BL/6 (B6) mouse intestine, an appreciable level of mouse Lpo gene expression was detected in the colon, but not the ileum. However, in B6 mice deficient in glutathione peroxidase (GPx)-1 and -2, GPx1/2-double-knockout (DKO), which had intestinal pathology, the colon Lpo mRNA levels increased 5- to 12-fold depending on mouse age. The Lpo mRNA levels in WT and DKO 129S1/SvlmJ (129) colon were even higher, 9- and 5-fold, than in B6 DKO colon. Higher levels of Lpo protein and enzymatic activity were also detected in the 129 mouse colon compared to B6 colon. Lpo protein was expressed in the differentiated colon epithelial cells, away from the crypt base, as shown by immunohistochemistry. Similar to human LPO mRNA, mouse Lpo mRNA had multiple spliced forms, although only the full-length variant 1 was translated. Higher methylation was found in the 129 than in the B6 strain, in DKO than in control colon, and in older than in juvenile mice. However, methylation of the Lpo intragenic CpG island was not directly induced by inflammation, because dextran sulfate sodium-induced colitis did not increase DNA methylation in B6 DKO colon. Also, Lpo DNA methylation is not correlated with gene expression.     

Demonstration of VIP receptors in B cells of the pancreas by quantitative ultrastructural autoradiography

Using quantitative electron microscopic autoradiography we demonstrated the existence of specific VIP receptors in B cells of the pancreatic islets. The presence of those receptors strongly suggests that the neuromediator can induce insulin release by direct stimulation of B cells.     

Long-term culture of epithelial cells from the normal rat colon

Summary Serial passage cultures of colonic epithelial cells from young rats have been maintained for more than 6 months in Eagle's minimum essential medium buffered with HEPES (25 mM) and supplemented with 2.5% fetal bovine serum, 0.5 μg/ml insulin, 5.0 μg/ml transferrin, and antibiotics. The cells proliferated in this medium with a population doubling time of approximately 53 h. The cells retained differentiated morphology as evidenced by secretory activity and the presence of secretory granules, microvilli, tonofilaments, and desmosomal junctions. Further cells at the fourth passage had normal karyotypes with 42 chromosomes and exhibited anchorage dependent growth. High concentrations of fetal bovine serum (10 to 15%) exerted toxic effects on the colonic epithelial cell cultures. Supported by National Cancer Institute Contract N01-CP-75914.     

Protection of human colon epithelial cells against deoxycholate by rottlerin

The bile salt, deoxycholate (DOC), can harm cells and cause disease. Hence, there is interest in identifying compounds capable of protecting cells against DOC. In HCT-116 colon epithelial cells, DOC increased generation of reactive oxygen species and caused DNA damage and apoptosis. These effects of DOC were inhibited by rottlerin, which is a phenolic compound of plant origin. In elucidating its mechansim, rottlerin prevented the release of cytochrome c from mitochondria into cytosol, and also prevented the cleavage of caspase-3. Yet, rottlerin by itself markedly decreased mitochondrial membrane potential and increased mitochondrial superoxide production, but this did not result in cytochrome c release or in caspase-3 cleavage. At a higher test concentration, two other phenolic phytochemicals, namely, quercetin and resveratrol, were each able to largely prevent the occurrence of apoptosis in cells exposed to DOC. In contrast, epigallocatechin gallate, curcumin, and genistein were ineffective.     

Activation of NADPH oxidase 1 in tumour colon epithelial cells

In the plasma membrane fraction from Caco-2 human colon carcinoma cells, active Nox1 (NADPH oxidase 1) endogenously co-localizes with its regulatory components p22(phox), NOXO1, NOXA1 and Rac1. NADPH-specific superoxide generating activity was reduced by 80% in the presence of either a flavoenzyme inhibitor DPI (diphenyleneiodonium) or NADP(+). The plasma membranes from PMA-stimulated cells showed an increased amount of Rac1 (19.6 pmol/mg), as compared with the membranes from unstimulated Caco-2 cells (15.1 pmol/mg), but other components did not change before and after the stimulation by PMA. Spectrophotometric analysis found approx. 36 pmol of FAD and 43 pmol of haem per mg of membrane and the turnover of superoxide generation in a cell-free system consisting of the membrane and FAD was 10 mol/s per mol of haem. When the constitutively active form of Rac, Rac1(Q61L) or GTP-bound Rac1 was added exogenously to the membrane, O(2)(-)-producing activity was enhanced up to 1.5-fold above the basal level, but GDP-loaded Rac1 did not affect superoxide-generating kinetics. A fusion protein [NOXA1N-Rac1(Q61L)] between truncated NOXA1(1-211) and Rac1-(Q61L) exhibited a 6-fold increase of the basal Nox1 activity, but NOXO1N(1-292) [C-terminal truncated NOXO1(1-292)] alone showed little effect on the activity. The activated forms of Rac1 and NOXA1 are essentially involved in Nox1 activation and their interactions might be responsible for regulating the O(2)(-)-producing activity in Caco-2 cells.     

Uptake of 3H-dopamine in megakaryocytes and blood platelets measured by quantitative electron-microscope autoradiography

Summary We studied the uptake of dopamine by mature megakaryocytes and blood platelets in mouse spleen after a single intraperitoneal injection of ³H-dopamine. In order to compare the uptake of ³H-dopamine in mature megakaryocytes and blood platelets, we used quantitative autoradiography at the electron-microscope level. Dense accumulations of silver grains were observed on both mature megakaryocytes and blood platelets; all other tissue elements of the spleen exhibited considerably less dense labeling. No sigificant differences with regard to dopamine uptake were observed in megakaryocytes and blood platelets. This is in contrast to the previous finding of very different patterns of ³H-5-hydroxytryptamine labeling in mature megakaryocytes and blood platelets (Daimon and Uchida 1985). The results of the present study provide new evidence in favor of the hypothesis that the active uptake mechanism of dopamine through the plasma membrane is different from the uptake mechanism of 5-hydroxytryptamine.     

Uptake of 3H-dopamine in megakaryocytes and blood platelets measured by quantitative electron-microscope autoradiography

We studied the uptake of dopamine by mature megakaryocytes and blood platelets in mouse spleen after a single intraperitoneal injection of 3H-dopamine. In order to compare the uptake of 3H-dopamine in mature megakaryocytes and blood platelets, we used quantitative autoradiography at the electron-microscope level. Dense accumulations of silver grains were observed on both mature megakaryocytes and blood platelets; all other tissue elements of the spleen exhibited considerably less dense labeling. No significant differences with regard to dopamine uptake were observed in megakaryocytes and blood platelets. This is in contrast to the previous finding of very different patterns of 3H-5-hydroxytryptamine labeling in mature megakaryocytes and blood platelets (Daimon and Uchida 1985). The results of the present study provide new evidence in favor of the hypothesis that the active uptake mechanism of dopamine through the plasma membrane is different from the uptake mechanism of 5-hydroxytryptamine.     

Deoxycholate induces DNA damage and apoptosis in human colon epithelial cells expressing either mutant or wild-type p53

Diets rich in fat result in higher concentrations of secondary bile acids or their salts in the colon, which may adversely affect cells of the colonic epithelium. Because secondary bile acids are thought to be genotoxic, exposing colon epithelial cells to secondary bile acids may induce DNA damage that might lead to apoptosis. The requirement for the p53 tumor suppressor gene in such events is unknown. In particular, the effects of secondary bile acids on colon epithelial cells having different p53 tumor suppressor gene status have not been examined. Therefore, HCT-116 and HCT-15 human colon adenocarcinoma cells, which express the wild-type and mutant p53 genes, respectively, were exposed to physiological concentrations of deoxycholate. The cells were then analyzed for evidence of DNA damage and apoptosis. After 2 h of incubation with 300 microM deoxycholate, both cell lines had greater levels of single-strand breaks in DNA as assessed by the comet assay. After 6 h of exposure to deoxycholate, HCT-116 and HCT-15 cells showed morphological signs of apoptosis, i.e., membrane blebbing and the presence of apoptotic bodies. Chromatin condensation and fragmentation were also seen after staining DNA with 4',6-diamidino-2-phenylindole. Other apoptotic assays revealed greater binding of annexin V-fluorescein isothiocyanate, as well as greater post-enzymatic labeling with dUTP-fluorescein isothiocyanate, by both cell lines exposed to deoxycholate. These data suggest that deoxycholate caused DNA damage in colon epithelial cells that was sufficient to trigger apoptosis in a p53-independent manner.     

Establishment of a long-term culture system for rat colon epithelial cells

Summary The aim of this study was to establish a long-term culture system for rat colon epithelial cells. Colonic crypts were isolated by incubating a 4-cm-long rat colon segment cut longitudinally with an ethylenediaminetetraacetic acid [disodium salts]-containing buffer, taken up in conditioned medium from the normal rat kidney fibroblast cell line NRK (i.e., the supernatant of pure NRK cultures), directly plated on mitomycin C-treated NRK cells and subcultured with conditioned medium from NRK cells. Cells started to migrate out of the crypts shortly after plating them on NRK feeder layers. Some of the crypts fell apart during the isolation procedure, whereas the vast majority of them did it within 1 to 2 h after plating. The cells proliferated extremely slowly but continuously over a period of 4 mo and were epithelial because they expressed cytokeratin 19 and were stained by crystal violet at pH 2.8. In conclusion, the experimental system described in this study allows to maintain rat colon epithelial cells for up to 4 mo in culture and can be used to study the effects of a variety of tumor-modulating factors on growth and gene expression of normal colon epithelial cells in vitro.     

Identification and characterization of a diamine exporter in colon epithelial cells

SLC3A2, a member of the solute carrier family, was identified by proteomics methods as a component of a transporter capable of exporting the diamine putrescine in the Chinese hamster ovary (CHO) cells selected for resistance to growth inhibition by high exogenous concentrations of putrescine. Putrescine transport was increased in inverted plasma membrane vesicles prepared from cells resistant to growth inhibition by putrescine compared with transport in inverted vesicles prepared from non-selected cells. Knockdown of SLC3A2 in human cells, using short hairpin RNA, caused an increase in putrescine uptake and a decrease in arginine uptake activity. SLC3A2 knockdown cells accumulated higher polyamine levels and grew faster than control cells. The growth of SLC3A2 knockdown cells was inhibited by high concentrations of putrescine. Knockdown of SLC3A2 reduced export of polyamines from cells. Expression of SLC3A2 was suppressed in human HCT116 colon cancer cells, which have an activated K-RAS, compared with their isogenic clone, Hkh2 cells, which lack an activated K-RAS allele. Spermidine/spermine N(1)-acetyltransferase (SAT1) was co-immunoprecipitated by an anti-SLC3A2 antibody as was SLC3A2 with an anti-SAT1 antibody. SLC3A2 and SAT1 colocalized on the plasma membrane. These data provide the first molecular characterization of a polyamine exporter in animal cells and indicate that the diamine putrescine is exported by an arginine transporter containing SLC3A2, whose expression is negatively regulated by K-RAS. The interaction between SLC3A2 and SAT1 suggests that these proteins may facilitate excretion of acetylated polyamines.     

Growth and characterisation of primary bovine colon epithelial cells in vitro

Epithelial crypts from the bovine colon were obtained by using a combined mechanical and enzymatic isolation method, followed by differential D-sorbitol gradient centrifugation. By using this isolation technique, a pure fraction of epithelial crypts with minimal mesenchymal contamination was obtained. The crypts were seeded in collagen-coated plastic flasks. The attached epithelial cells proliferated and formed a confluent monolayer after 6 days in culture. Under low-serum culture conditions (1% fetal calf serum), the cells had a population doubling time of 21-22 hours. During the culture period, the colonocytes were characterised morphologically and enzymatically. The morphology of the cultured cells was confirmed by scanning electron microscopy and transmission electron microscopy. The presence of microvilli, tight junctions and desmosomes demonstrated the ability of the cultured cells to restore an epithelial-like cell monolayer. The epithelial origin of the cells was demonstrated by labelling the cells with antibodies against epithelial-specific cytokeratins 7 and 13. The functional integrity of the cells was evaluated by measuring various marker enzymes (gamma-glutamyltranspeptidase, acid phosphatase, alkaline phosphatase, NADH-dehydrogenase) and membrane-associated Na+-K+-ATPase activity. Membrane integrity was determined by measuring the leakage of lactate dehydrogenase into the culture medium. This new culture system for bovine colon epithelial cells could be used as an in vitro model of the colon epithelium in physiological and toxicological studies.