Parental HT29 Cells Research Articles

Expressed sequence tag (EST) phenotyping of HT-29 cells: cloning of ser/thr protein kinase EMK1, kinesin KIF3B, and of transcripts that include Alu repeated elements

To study the mechanisms that control epithelial commitment and differentiation we have used undifferentiated HT-29 colon cancer cells and a subpopulation of mucus secreting cells obtained by selection of HT-29 cells in 10 −6 M methotrexate (M6 cells) as experimental models. We isolated cDNAs encoding transcripts overexpressed in early confluent M6 cells regarding steady-state levels in HT-29 cells by subtractive hybridisation. Fifty-one cDNA clones, corresponding to 34 independent transcripts, were isolated, partially sequenced by their 5′ end, and classified into four groups according to their identity: transcripts that included a repeated sequence of the Alu family (10 clones, among them those encoding ribonucleoprotein RNP-L and E-cadherin), transcripts encoded by the mitochondrial genome (nine clones), transcripts encoding components of the protein synthesis machinery (23 clones, including the human ribosomal protein L38 not previously cloned in humans) and nine additional cDNAs that could not be classified in the previous groups. These last included ferritin, cytokeratin 18, translationally controlled human tumour protein (TCHTP), mt-aldehyde dehydrogenase, as well as unknown transcripts (three clones), and the human homologues of the molecular motor kinesin KIF3B and of the ser/thr protein kinase EMK1. Spot dot and Northern blot analyses showed that ser/thr protein kinase EMK1 was differentially expressed in M6 cells when compared with parental HT-29 cells. Steady-state levels of EMK1 were higher in proliferating, preconfluent, M6 and HT-29 cells than in 2 days post confluence (dpc) and 8dpc M6 and HT-29 cells. Transcripts that included an Alu repeat were also shown to be differentially expressed and accumulated in differentiating M6 cells when analysed by Northern blot. The significance of the transcripts cloned is discussed in the context of the commitment and differentiation of the M6 cells to the mucus secreting lineage of epithelial cells.

Alteration in mucin gene expression and biological properties of HT29 colon cancer cell subpopulations.

Previous studies from our laboratory have shown that HT29 cells selected by adaptation to methotrexate (HT29-MTX) express mature mucins that differ in their immunoreactivity to antibodies against gastric mucin and in the level of one of two major gastric mucin MUC5AC (MUC5) mRNA compared with parental HT29 cells. In this study, we examined the expression of another major gastric mucin, MUC6 mRNA, as well as that of MUC2, -3 and -5 mRNAs in HT29-MTX cells. We also examined their relationship to mucin-related antigen expression and biological properties of the cells such as adhesion to matrigel and E-selectin and in vitro invasiveness, liver colonising activity and degree of differentiation of nude mouse xenograft. Slot blot and Northern analysis revealed markedly increased levels of MUC5 mRNA but no change in MUC6 mRNA level in HT29-MTX cells compared with parental HT29 cells which express barely detectable levels of MUC6 mRNA. A nuclear run-on study showed that MUC5 mRNA was up-regulated at the transcriptional level. The marked increase in MUC5 mRNA was associated with a significant increase in the expression of human gastric mucin and apomucin antigens in HT29-MTX cells. When the adhesive capacity of two cell lines was compared, HT29-MTX cells showed significantly lower adhesion to E-selectin consistent with their lower expression of sialyl Le(x) and sialyl Le(a) antigens compared with HT29 cells. HT29-MTX cells also showed lower adhesive capacity to matrigel than HT29 cells. Interestingly, HT29-MTX cells exhibited significantly decreased liver colonisation capacity in nude mice following splenic vein injection. Furthermore, nude mouse xenograft tumours produced by HT29-MTX cells exhibited a significantly greater degree of differentiation, consisting of mucin-secreting glands than those produced by HT29 cells. In conclusion, these results indicate a shift of predominantly colonic-type mucins to the gastric type, specifically the surface epithelial cell type (MUC5) but not the mucous neck cell or antral gland type (MUC6) in HT29-MTX cells and strongly suggest that altered regulation of mucin genes and the degree of differentiation in cancer cells may be responsible for the altered biological behaviour of these cells.

Identification of a 42-kDa Nuclear Factor (NF1-MUC5B) from HT-29 MTX Cells That Binds to the 3′ Region of Human Mucin GeneMUC5B

MUC5B gene is one of the four human mucin genes mapped to chromosome 11p15. The identification of three potential Sp1 binding sites located between the tandem repeat and the 3′ end ofMUC5Bsuggests a possible regulatory role for this region. In this report we show by electrophoretic mobility shift assay that only one potential Sp1 binding site (NAU62) leads to a specific interaction with a nuclear factor from HT-29 MTX cells which does not exist in parental HT-29 cells. By using mutated versions of NAU62, an 18 mer sequence within this later was shown to be directly involved in the interaction. The nuclear factor called NF1-MUC5B which binds to this element has a Mrof 42000 and is not Sp1. These results suggest thatMUC5Bcontains a sequence in its 3′ region that might act as a cis-element. This report opens the field of transcriptional regulation of human mucin genes encoding secreted mucins.

Differential expression of the human mucin genes MUC1 to MUC5 in relation to growth and differentiation of different mucus-secreting HT-29 cell subpopulations.

Mucin expression was analysed, in relation to cell growth, in parental HT-29 cells and in two populations of mucus-secreting HT-29 cells selected by adaptation to methotrexate (HT29-MTX) or 5-fluorouracil (HT29-FU). These two populations express mature mucins that differ in their immunoreactivity to antibodies against gastric (HT29-MTX) or colonic mucins (HT29-FU). In the parental population, at late confluency, only very few cells produce mucins or the MUC1 glycoprotein, this being consistent with the low level of expression of the mRNAs corresponding to the MUC1 to MUC5C mucin genes. In the HT29-MTX and HT29-FU populations, the appearance of mucus droplets, as shown by histochemistry and immunofluorescence, starts a few days after confluency, progressively involving a greater proportion of cells and reaching a steady state at late confluency. The MUC1 glycoprotein appears earlier, already being detectable in preconfluent cells. Its distribution is restricted to the apical surface of the cells and is distinct from that of the mucus droplets. In both populations the growth-related levels of MUC1 mRNA are concordant with the apparent levels of expression of the MUC1 glycoprotein. The levels of MUC2, MUC3, MUC4 and MUC5C mRNAs differ from one population to another and, within each population, according to the stage of the culture. The highest levels of MUC2 and MUC4 mRNAs are found in the HT29-FU cells, whereas the highest levels of MUC3 and MUC5C are found in the HT29-MTX cells, suggesting that the differences observed in the mature mucins expressed by either population may be related to which MUC genes are expressed. In both populations significant or even high levels of MUC mRNAs are already present in early cultures, i.e. at a stage when the mature mucins are not yet detectable, suggesting that mucin maturation is a later event.

CHROMOSOMAL ALTERATIONS IN PERMANENTLY DIFFERENTIATED HT29 COLON-CARCINOMA CELLS INDUCED WITH SODIUM-BUTYRATE

We have established differentiated subclones from sodium butyrate-treated HT29 colon carcinoma cells. Two subclones, CIII and CVII, grew as tightly-compacted colonies and showed polarized confluent monolayers in vitro. CIII cells formed domes in vitro, a morphological characteristic of columnar absorptive epithelium. Besides these differentiated characteristics, CIII and CVII cells showed lower tumorigenicity than parental HT29 cells. Karyotypic analysis indicated that all cells retained chromosomes in the triploid range. However, modal chromosome numbers among 30 metaphases of each clone were concomitantly reduced from 71 (in HT29) to 68 (in CVII) or 67 (in CHI) with the differentiated phenotypes. Our preliminary karyotype analysis has suggested that the number of chromosomes 10q21-qter, 14q22-qter and 16q were decreased in both subclones. To confirm this, DNAs from these cells and another butyrate-induced subclone, CI-1, were analyzed with several DNA markers. Southern blot analysis indicated that parental HT29 cells were polymorphic for the BamHI and TaqI RFLPs detected by urokinase (7 kbp and 1.6 kbp) and D10S25 (2.5 kbp and 2.1 kbp) probes, respectively, both of which are located on chromosome band 10q. In CI-1 and CVII cells, allelic losses of urokinase (1.6 kbp) and D10S25 (2.5 kbp) were observed, respectively. Although both alleles of these markers were conserved in CIII cells, one allele of urokinase (7 kbp) and D10S25 (2.1 kbp) was less representative. Southern blot analysis using probes for Fos proto-oncogene, D14S20 marker type IV collagenase gene, haptoglobin gene located on chromosome bands 14q and 16q did not show any loss or rearrangement occurred in these subclones. Similarly, no loss or rearrangement was observed for the krev-1 gene on chromosome 1, whose number was not changed in either subclone. These findings indicate that in CI- 1, CIII, and CVII cells, the number of chromosome band 10q is decreased relative to HT29 cells. These deletions may be implicated in the morphological differentiation of HT29 cells treated with sodium butyrate.

Transfer of the IL-6 gene into a human colorectal carcinoma cell line and consequent enhancement of tumor antigen expression

cDNA encoding the human IL gene (580 bp), inserted into a retroviral expression vector carrying neomycin resistance selective marker, was introduced into HT-29 human colon carcinoma cells by lipofection. Interleukin-6 activity was measured by ELISA and bioassay using B9 cells. Interleukin-6 secreted by transfected HT-29 cells was shown to be biologically active. The expression of the human tumor associated antigen CEA (carcinoembryonic antigen), HLA classes I and II, and ICAM-1 antigens in the transfected HT-29 cells were also analyzed by flow cytometry. Significant enhancement in the expression of CEA but not in the expression of HLA class I, HLA class II and ICAM-1 antigens, was observed in the transfected HT-29 cells as compared to the parental HT-29 cells. These results provide experimental evidence that enhancement of tumor antigen expression on tumor cells can be induced by IL-6 gene transfection, and suggest another potential role for the use of IL-6 gene transfer in the immunotherapy of human cancers.

Analysis of the relationship between stage of differentiation and nk/lak susceptibility of colon carcinoma cells

NK and LAK cells which are able to lyse tumor target cells in an MHC-unrestricted manner are not equally effective against targets of the same nature. In the case of colorectal tumors, some cells are highly sensitive, whereas others are resistant to NK and can even be quite resistant to LAK-mediated lysis. In the present paper, we tried to correlate the stage of differentiation of 17 human colorectal tumor cell lines with their NK- or LAK-cell susceptibility. It was observed that NK cells killed colorectal target cells independently from their stage of differentiation defined according to histopathological criteria from xenografting in nude mice. NK susceptibility was not correlated either with in vitro-defined criteria of differentiation, such as cell polarity and morphology, brush-border enzyme expression and CEA production. A LAK-resistant HT-29 sub-line (HT-29 LAK) was selected which could not be distinguished from HT-29 in terms of features of differentiation. It was further observed that HT-29 Glc-/+ cell line, a highly differentiated enterocytic-like variant of HT-29, obtained after glucose starvation, was killed by LAK cells as efficiently as the moderately differentiated parental HT-29, and that Caco-2 cells, which differentiate spontaneously after confluence in standard culture conditions, were equally sensitive to NK-mediated lysis whatever their stage of differentiation. In contrast, HT29 MTX10(-5), a highly differentiated mucus-secreting variant of HT29 obtained by methotrexate selection, was much more resistant to LAK cells than parental HT29 cells.

Expression of receptors for enterotoxigenic Escherichia coli during enterocytic differentiation of human polarized intestinal epithelial cells in culture.

To study the expression of human intestinal receptors for enterotoxigenic Escherichia coli (ETEC), the human polarized intestinal epithelial cell line Caco-2 in culture and several subpopulations of HT-29 cells in culture--parental (mainly undifferentiated) HT-29 cells (HT-29 Std), an enterocytelike subpopulation obtained by selection through glucose deprivation (HT-29 Glc-), and an enterocytelike subpopulation obtained by selection through glucose deprivation which maintains its differentiation characteristics when switched back to standard glucose-containing medium (HT-29 Glc-/+)--were used. Since Caco-2 spontaneously differentiated in culture under standard culture conditions (in the presence of glucose) and HT-29 cells were undifferentiated when cultured under standard conditions (HT-29 Std) and differentiated when grown in a glucose-free medium (HT-29 Glc-), we studied the expression of the receptors for colonization factor antigens (CFA) I, II, and III and the 2230 antigen of ETEC in relation to enterocytic differentiation. We provide evidence that expression of ETEC CFA receptors develops in parallel with other differentiation functions of the cultured cells. The expression of ETEC-specific brush border receptors was studied by indirect immunofluorescence using antibodies raised against purified ETEC CFA. No ETEC receptors were detected in HT-29 Std or short-term-cultured Caco-2 cells. However, among the population of HT-29 Std cells, 2 to 4% of the cells were found to bind ETEC, and these cells expressed positive carcinoembryonic antigen immunoreactivity. This indicated that among the population of undifferentiated HT-29 cells, clusters of differentiated cells were present. ETEC CFA receptors were expressed in the apical and basolateral domains of differentiated HT-29 cells, whereas in differentiated Caco-2 cells only apical expression was observed. Both in HT-29 cells (HT-29 Glc-/+) and in Caco-2 cells cultured under standard conditions, ETEC CFA receptors develop as a function of day in culture. This indicated that the expression of the ETEC CFA receptors was a growth-related event. Indeed, ETEC CFA receptors developed in step with the apical expression of differentiation-associated proteins.