Growth Inhibition Of HT-29 Cells Research Articles

Induction of retinoic acid receptor β mediates growth inhibition in retinoid-resistant human colon carcinoma cells

The molecular mechanisms underlying the differential sensitivity of human colon carcinoma cells to retinoid mediated growth inhibition are poorly understood.To identify the intracellular mechanisms responsible for resistance against retinoid mediated growth inhibition in human colon carcinoma cells.Anchorage independent growth of the human colon carcinoma cell lines HT29 and LoVo was determined by a human tumour clonogenic assay. Retinoid receptor expression was evaluated by reverse transcription polymerase chain reaction and northern blotting. Retinoid mediated transactivation was assessed by transient transfection of a pTK::betaREx2-luc reporter construct. Retinoid receptor overexpression was achieved by selecting stably transfected cell clones.Retinoid treatment resulted in profound dose dependent growth inhibition in HT29 cells, while LoVo cells were unaffected. The two cell lines express identical patterns of nuclear retinoid receptor mRNA transcripts. However, on retinoid treatment, retinoic acid receptor beta gene expression was upregulated only in retinoid sensitive HT29 cells, but not in retinoid resistant LoVo cells. In accordance, stable overexpression of retinoic acid receptor beta but not alpha or gamma conferred retinoid mediated growth inhibition on LoVo cells.Induction of retinoic acid receptor beta expression is required and sufficent to confer retinoid mediated growth inhibition on human colon carcinoma cells.

Expression of proliferation‐associated nuclear autoantigens, p330d/CENP‐F and PCNA, in differentiation and in drug‐induced growth inhibition using two‐parameter flow cytometry

p330d/CENP-F is a recently described nuclear autoantigen that was detected in PHA-stimulated but not in resting peripheral lymphocytes. This protein accumulates in the nucleus during S-phase and reaches maximum levels during the G2 and M phases of the cell cycles. We compared the expression of p330d/CENP-F and proliferating cell nuclear antigen (PCNA) during the induction of terminal myeloid differentiation of HL-60 tumour cells. HL-60 cells were induced to differentiate with retinoic acid (RA), dimethyl sulfoxide (DMSO), and 3-nitrobenzothiazolo [3,2-]quinolinium (NBQ), and collected at different intervals. Control and treated cells were analyzed by two-parameter flow cytometry using propidium iodide and antibodies to p330d/CENP-F and PCNA. The percentage of p330d/CENP-F and PCNA positive cells was found to be proportional to the percentage of proliferating cells. After two cell cycles (65 h), the percentage of p330d/CENP-F and PCNA positive cells was reduced proportionately to the number of cells that had differentiated. Reduction in the expression of both antigens was completed after 120 h when 80% to 85% of the cells were arrested in G1 and displayed the mature phenotype. The expression of p330d/CENP-F and PCNA was also assessed in the growth inhibition of HT-29 cells induced by various concentrations of camptothecin (CPT), etoposide (VP-16), and aphidicolin (APH). There was a dose-dependent displacement of cells to late S-phase by CPT while VP-16 induced cells to accumulate in G2+M, and as expected these effects caused a strong increase in the cellular levels of both antigens. The arrest of cells in G1 by APH led to a significant decrease in their expression. The dramatic reduction in p330d/CENP-F levels during differentiation, and the correlation of its expression with the cell cycle effects of the cytotoxic drugs are consistent with the behaviour expected for a proliferation marker.

Vitamin D and its metabolites inhibit cell proliferation in human rectal mucosa and a colon cancer cell line.

Like calcium, vitamin D may protect against colorectal neoplasia as it reduces epithelial cell proliferation and induces differentiation. Although its therapeutic use is limited by its effects on calcium metabolism, analogues such as calcipotriol produce little hypercalcaemia. Stathmokinetic and immunohistochemical techniques were used to study the effect of 1,25 (OH)2 D3 and its analogues on cell proliferation in human rectal mucosa and a colon cancer cell line. Paired sigmoidoscopic biopsy specimens were obtained from 17 control patients and five patients with familial adenomatous polyposis. Explants were established in organ culture, with or without the addition of vitamin D. Proliferation was assessed using (1) metaphase arrest to determine the crypt cell production rate (CCPR) and (2) Ki-67 monoclonal antibody directed against an antigen present in proliferating cells. 1,25 (OH)2 D3 in concentrations of 1 microM-100 pM (10(-6)-10(-10) M) reduced the CCPR (cells/crypt/hour) from 4.74 to 2.15-2.67 (p < 0.001), and the Ki-67 labelling index from 7.28-3.74 (p < 0.01). Likewise, vitamin D2, 10 nM (10(-8) M) reduced the CCPR from 4.74-2.74 (p < 0.05) and calcipotriol from 4.86-2.38 (p < 0.05). In familial adenomatous polyposis patients 1,25 (OH)2 D3 100 pM (10(-10) M) halved the CCPR from 8.75-4.22. Calcipotriol (10(-5) M to 10(-9) M) produced a clearcut dose response inhibition of HT-29 cell growth. Thus, vitamin D and its metabolites inhibit proliferation in normal and premalignant rectal epithelium and suppress growth in a colorectal cancer cell line.

Inhibition of ribonucleotide reductase and growth of human colon carcinoma HT-29 cells and mouse leukemia L1210 cells by N-hydroxy- N′-aminoguanidine derivatives

A series of N-hydroxy- N′-aminoguanidine (HAG) derivatives were studied and compared for their effects on ribonucleotide reductase activity in cell-free extracts; on nucleic acid synthesis and the growth of human colon carcinoma HT-29 cells; and on mouse leukemia L1210 cells in culture. The HAG derivatives [RCHNNHC(NH)NHOH-tosylate] studied could be grouped as; (1) hydroxy-benzylidines; (2) methoxybenzylidines; and (3) nitrobenzylidines substituted at the R position. 2′-Hydroxybenzylidine-HAG, the lead compound, was relatively active in both HT-29 cells and L1210 cells (20 ± 5 and 13 ± 4 μM for 50% inhibition of HT-29 and L1210 cell growth respectively). The monohydroxybenzylidene compounds were generally more active than the dihydroxy- and trihy-droxybenzylidene-HAG derivatives. The methoxybenzylidene-HAGs were as active as the monohy-droxybenzylidene-HAGs. 2′-Hydroxy-4′-methoxybenzylidene-HAG was much more active than 2′,4′-dihydroxybenzylidene-HAG. The mononitrobenzylidene-HAGs were more active than the dinitro-benzylidene-HAG compound. In general, L1210 cells were more sensitive to the effects of the HAG compounds than were HT-29 cells. There was good agreement between the concentration of drug required to inhibit the growth of HT-29 cells and that required to inhibit the growth of L1210 cells. There was also good correlation between the ability of HAG derivatives to inhibit ribonucleotide reductase activity and to inhibit tumor cell growth. Some derivatives, such as 2′,3′,4′- and 3′,4′,5′-trihydroxybenzylidene-HAG inhibited L1210 cell growth by 50% at lower concentrations (7.8 and 11.9 μM respectively) than the concentrations needed for 50% inhibition of HT-29 cell growth (196 and 234 μM respectively) and ribonucleotide reductase activity (122 and 188 μM respectively). The studies of nucleic acid synthesis in L1210 cells using [ 3H]cytidine as a precursor showed that 2′,3′,4′-trihy-droxybenzylidine-HAG inhibited DNA synthesis at a lower concentration (29 μM for 50% inhibition) than was needed for the inhibition of RNA synthesis and formation of [ 3H]deoxycytidine nucleotides in the acid-soluble fraction (320 and 820 μM for 50% inhibition respectively). These results indicate that 2′,3′,4′-trihydroxybenzylidine-HAG inhibits DNA synthesis in L1210 cells through other mechanisms rather than exclusively through the inhibition of ribonucleotide reductase activity.

Effect of ribonucleotide reductase inhibitors on the growth of human colon carcinoma HT-29 cells in culture

The effects of ribonucleotide reductase inhibitors on the growth of the human colon carcinoma cell line HT-29 were examined. Inhibitors were chosen for these studies that were specifically directed at each of the subunits of ribonucleotide reductase. The concentrations of drugs required to inhibit the growth of HT-29 cells by 50% (IC50) for hydroxyurea, 2,3-dihydro-lH-pyrazole-[2,3a]imidazole (IMPY), and 4-methyl-5-amino-l-formyl-isoquinoline thiosemicarbazone (MAIQ) were 206, 996, and 3.2 microM, respectively. Although the IC50 for deoxyadenosine alone was greater than 2,000 microM, in the presence of 5 microM erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), which protects deoxyadenosine from deamination by adenosine deaminase, it was reduced to 112 microM. The IC50 for deoxyguanosine was 1,060 microM. The addition of 8-aminoguanosine to protect deoxyguanosine from phosphorolysis by purine nucleoside phosphorylase did not increase the toxicity of deoxyguanosine in HT-29 cells. The combination of MAIQ or IMPY and deoxyadenosine/EHNA gave strong synergistic inhibition of HT-29 cell growth. The results of these studies indicate that ribonucleotide reductase inhibitors effectively block the growth of human colon carcinoma HT-29 cells and that combinations of inhibitors directed at the individual subunits of reductase result in synergistic inhibition of HT-29 cell growth in culture.