Inhibition of IL-12 production by 1,25-dihydroxyvitamin D3. Involvement of NF-kappaB downregulation in transcriptional repression of the p40 gene.

Interleukin 12 (IL-12), produced by myelomonocytic cells, plays a pivotal role in the development of T helper 1 (Th1) cells, which are involved in the pathogenesis of chronic inflammatory autoimmune disorders. 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] inhibits IL-12 production by activated macrophages and dendritic cells, thus providing a novel interpretation to its immunosuppressive properties. 1,25(OH)2D3 significantly inhibits mRNA expression for both IL-12 p35 and p40 subunits acting at the transcriptional level. The effect of 1,25(OH)2D3 on p40 promoter activation was analyzed by cotransfecting monocytic RAW264.7 cells with p40 promoter/reporter constructs and expression vectors for vitamin D3 receptor (VDR) and/or retinoid X receptor (RXRalpha). We observed transcriptional repression of the p40 gene by 1,25(OH)2D3, which required coexpression of VDR with RXR and an intact VDR DNA-binding domain. The repressive effect maps to a region in the p40 promoter containing a binding site for NF-kappaB (p40-kappaB). Deletion of the p40-kappaB site abrogates part of the inhibitory effect on the p40 promoter, confirming the functional relevance of this site. Activation of monocytic THP-1 cells in the presence of 1,25(OH)2D3 results in reduced binding to the p40-kappaB site. Thus, 1,25(OH)2D3 may negatively regulate IL-12 production by downregulation of NF-kappaB activation and binding to the p40-kappaB sequence.

Regulation of progesterone receptor gene expression.

The levels of progesterone receptor (PR) mRNA, PR protein, and [3H]R5020 binding activity were measured in parallel experiments conducted on a T47D subline expressing the estrogen receptor. A significant increase of PR mRNA levels could be detected within 6 h of exposure of the cells to estradiol (10(-8) M). The changes in mRNA, however, did not lead to any variation of PR protein levels of [3H]R5020 binding activity. A parallel analysis of PR mRNA and [3H]R5020 binding was then performed in a series of tumor biopsies. In estrogen receptor-positive and PR-positive tissues a correlation among the two values was found. It is postulated that the above mentioned data could reflect the existence of a difference in the mechanisms controlling the numerous steps of the PR synthesis in the various hormone-responsive tissues. This variability could allow an organ-specific response to the cyclic changes of circulating hormone.

Different mechanisms regulate the IFN-inducible expression of 40 kDa human 2'-5' oligo (A) synthetase in HeLa and Molt 4 cells.

Expression of 40 kDa 2'-5' oligo (A) synthetase can serve as a marker of interferon (IFN) activity on the biological target. The mechanisms of induction of human 40 kDa 2'-5' oligo (A) synthetase by IFNs were investigated in HeLa and Molt 4 cells. Using a combined treatment with cycloheximide and actinomycin D we observed that in HeLa cells IFN-alpha did not need ongoing protein synthesis to induce the enzyme, whereas the addition of cycloheximide prevented the induction by IFN-gamma. IFN-alpha induced the 40 kD enzyme in the T-cell line Molt 4 to a level comparable to that in HeLa cells, but only in the presence of active protein synthesis. These results suggest that an early response gene coding for a positive IFN-inducible protein may be needed in T cells, but not in HeLa cells to regulate the transcription of this 2'-5' oligo (A) synthetase gene.

Effects of cholesterol and alfa-2-recombinant interferon on cell growth and cell antigen production in the HT 29 cells, an established cell line of human colon adenocarcinoma.

HT 29 cells, an established cell line of human colon adenocarcinoma, were grown in RPMI 1640 medium without or with cholesterol at 25, 50, 100 micrograms/ml concentrations. In some experiments 100 or 200 U/ml alfa-2-A recombinant Interferon were added to the medium. Only in the case of the highest cholesterol concentration there was a reduced number of cells at confluence. Moreover, only the production of CEA increased in the presence of cholesterol. Interferon did not affect cell growth appreciably but stimulated CEA release into the medium during the first three days of culture. Morphological analysis of cells in the presence of cholesterol seems to indicate an attempt of the cells to differentiate.

Production of polyclonal antibodies against the 40 kDa form of human 2'-5' oligoadenylate synthetase.

A 17-aminoacid peptide corresponding to the C terminal of the smaller form of human 2'-5' oligoadenylate synthetase was coupled to keyole lymphet haemocyanin (KLH) and used as immunogen in rabbits. After a cycle of four immunizations two animals produced immunoglobulins able to recognize the 17-aminoacid peptide as evaluated in ELISA assays. The specific Ig were purified by an immunoadsorbent with the peptide immobilized on Sepharose CL-4B and used in Western blot employing either protein A iodinated or conjugated with peroxidase as indicator system. The results obtained using extracts from HeLa or WISH cells treated for 15 hr with HuIFN-alfa as antigen demonstrate that the anti-peptide antibodies recognize the 40 kDa form of the 2'-5' oligoadenylate synthetase enzyme complex. These antibodies therefore represent a useful tool for monitoring the induction of the above enzyme.

Effects of alpha, beta and gamma recombinant interferons on CEA production from HT-29 human colon adenocarcinoma cell line.

The human colon adenocarcinoma derived cell line HT-29 is a good in vitro model for the study of CEA production and release under various experimental conditions. Many studies indicate that CEA secretion is correlated with cell proliferation and seems to depend on the growth conditions and differentiation characteristics induced by the culture medium. The present study demonstrates that recombinant interferons alpha, beta and gamma (rIFN alpha, rIFN beta, rIFN gamma) can modify CEA production and release by HT-29 cell-line. rIFN gamma in particular causes an enhancement of CEA production and release in the culture medium. This dose-depending effect is in some way correlated to cell growth inhibition since the enhancement of CEA expression in the interferon treated cells is evident in the presence of a reduction in cell proliferation. The activity of rIFN alpha and rIFN beta on CEA release is much less remarkable than that demonstrated by rIFN gamma, and is probably only due to the fact that HT-29 colon adenocarcinoma cells respond poorly to the effects of rIFN alpha and rIFN beta at the doses we used. These findings suggest that CEA production, expression and release can be modulated in a variety of ways under the influence of different rIFN treatment and this situation must be taken into account in immunodiagnostic and immunotherapeutic applications of anti-CEA monoclonal antibodies in the cancer patient.

Tissue polypeptide antigen and carcinoembryonic antigen in colon tumors: serum levels and immunohistochemical localization.

Patients with malignant and benign colon disease (59 colon cancer and 96 polyps) were studied by means of tissue polypeptide antigen (TPA) and carcinoembryonic antigen (CEA) tests. The evaluation of the circulating levels of the markers showed that the overall sensitivity for the TPA test was 57.6% and for the CEA test was 55.9%. Their specificities were 89.5% and 94.7%, respectively. The analysis of results indicated no considerable difference between CEA and TPA in detecting individuals with malignant diseases. There was only a slight difference in Dukes stages: in stages A and B, TPA sensitivity was higher than CEA sensitivity. On the contrary, in the group of patients with polyps, more false-positive results were obtained with the TPA test than with the CEA test. Immunohistochemical studies on the small group of patients (12 colon cancers) allowed us to evaluate the relationship between the staining positivity for the anti-TPA and anti-CEA antibodies and the circulating levels of the markers. The staining in some cases was not correlated either with the stage of cancer or the circulating TPA or CEA levels. This fact further shows the need to investigate the mechanism that determines the blood levels of many tumor markers. All the specimens examined were positive for TPA and CEA staining, but they were composed of varying proportion of positive and negative tumor cells. The degree of positivity was frequently variable not only between the specimens but also within the same specimen.