Histone deacetylase inhibitors increase p21(WAF1) and induce apoptosis of human myeloma cell lines independent of decreased IL-6 receptor expression.

Histone deacetylase (HDAC) inhibitors cause growth arrest and apoptosis of cancer cells by both p21-dependent and independent mechanisms. Decreased expression of growth factor receptors may be a key factor in the p21-independent mechanism, although this has not been directly tested. We have tested the effects of sodium butyrate and trichostatin A on human myeloma cell lines and have observed G1 arrest and apoptosis associated with increased expression of p21(WAF1), Bax, Rb dephosphorylation, and decreased IL-6 receptor (IL-6R) expression. Experiments to determine the role of disruption of IL-6 signaling as a result of decreased IL-6 receptor expression in mediating these effects were conducted using a stable transfectant of the OPM-2 line which constitutively expressed the IL-6 receptor. Our results indicated that decreased IL-6R expression was not required for induction of p21(WAF1) or apoptosis. Thus, HDAC inhibitors appear to activate multiple cellular pathways, leading to growth arrest and apoptosis, and their use in the treatment of myeloma, particularly in combination with other agents, warrants further investigation.

Growth inhibition of a human myeloma cell line by all-trans retinoic acid is not mediated through downregulation of interleukin-6 receptors but through upregulation of p21(WAF1).

All-trans retinoic acid (ATRA) has previously been shown to inhibit the growth of OPM-2 human myeloma cells. The growth inhibition was postulated to result from a transcriptional downregulation of interleukin-6 receptor alpha (IL-6Ralpha) with IL-6Rbeta (gp130) unaffected. To formally test this hypothesis, an expression vector designed for constitutive IL-6Ralpha expression was constructed and used for transfection of OPM-2 cells. Six stable transfectants were cloned. The expression of IL-6Ralpha was shown by immunofluorescence with anti-IL-6Ralpha antibody and 125I-IL-6 binding. In five of six transfectant clones, cellular IL-6Ralpha was 1.5- to 6-fold higher than the parental cells, with the ligand binding affinity unchanged. While ATRA reduced IL-6Ralpha expression in the parental OPM-2 cells, it enhanced its expression in these five transfectants. The clonogenic growth of these transfectants, however, remained strongly inhibited by ATRA. Further analysis, comparing the parental OPM-2 cells and a representative transfectant, clone C5, showed that IL-6 caused rapid tyrosine phosphorylation of gp130 in both OPM-2 and C5 clones. Pretreatment with ATRA greatly reduced IL-6-induced gp130 phosphorylation in OPM-2 cells, reflecting a reduction in cellular IL-6Ralpha. In contrast, IL-6-induced gp130 phosphorylation was not reduced by ATRA pretreatment in C5 cells, indicating that the expressed IL-6Ralpha was functional. Similar to OPM-2 cells, C5 cells were sensitive to growth inhibition by dexamethasone, which was entirely reversed by exogenous IL-6, suggesting that the IL-6 postreceptor signal transduction remained intact. ATRA was further shown to upregulate p21(WAF1) expression and cause dephosphorylation of the retinoblastoma protein (pRB) in both OPM-2 and C5 cells. Exogenous IL-6 also failed to reverse these effects of ATRA. Thus, the growth inhibitory activity of ATRA is not mediated through cellular IL-6Ralpha downregulation and is likely to result from a direct upregulation of p21(WAF1) and consequent dephosphorylation of pRB.

Inhibition of myeloma cell growth by all-trans retinoic acid is associated with upregulation of p21WAF1 and dephosphorylation of the retinoblastoma protein.

Retinoic acid and dexamethasone, in combination, inhibit the growth of human myeloma cell lines in a synergistic manner. Previously, we observed that all-trans retinoic acid (ATRA) caused G1 arrest and inhibited clonogenic growth of the OPM-2 human myeloma cell line. This was associated with downregulation of the IL-6 receptor (IL-6R) gp80 protein, while autocrine IL-6 production and gp130 were not affected. Growth inhibition was not reversed by the addition of exogenous IL-6 or forced, constitutive expression of the IL-6 receptor gp80 protein, suggesting that the mechanism of action of ATRA may be due to effects on the post-receptor pathway. Therefore, in this study we have investigated whether growth arrest was associated with changes in the level of phosphorylation of the RB protein. ATRA decreased the level of phosphorylation of the RB protein at doses > 5 x 10(-9) M and also induced a five fold increase in p21WAF1, while levels of p27KIP1 and CDK2 were unchanged. The ATRA-mediated increase in p21 preceded the change in RB phosphorylation and G1 arrest and was not reversed by the addition of exogenous IL-6. The levels of CDK2 activity were inhibited approximately 60% in ATRA-treated cells, suggesting that the increased p21 levels were sufficient to inhibit CDK activity and cause RB hypophosphorylation. Increased levels of p21 have recently been observed in human myeloma cells exposed to dexamethasone, and we suggest that the common ability of these two agents to inhibit myeloma cell growth depends on their induction of p21.

Urinary N1-acetylspermidine and N8-acetylspermidine excretion in normal humans and in patients with colorectal cancer.

Urinary N1-acetylspermidine (N1SPD) and N8-acetylspermidine (N8SPD) were measured in 24-hr urine specimens from 42 patients with colon adenocarcinoma and 29 healthy controls to assess their use as markers for colon cancer screening. Serial spot urines in four controls demonstrated significant fluctuations in these polyamine levels throughout the day without a distinct circadian pattern and therefore all subsequent analyses were performed on 24-hr collections. Both N1SPD and N8SPD were significantly increased in colon cancer patients compared to controls. Neither test correlated with tumor stage or location, but N8SPD was elevated in patients with poorly differentiated adenocarcinoma when compared to moderate or well-differentiated tumors. Using receiver operator characteristic (ROC) analysis, N1SPD had a higher information content than N8SPD, N1SPD + N8SPD, or the ratio of N1SPD/N8SPD and at a normal cut-off value of 4.0 nmol/mg creatinine, yielded a 95% specificity and 50% sensitivity for colon cancer.