Analysis of historical negative control group data from the rat in vivo micronucleus assay.

A database of micronuclei counts for historical negative control data from rat in vivo micronuclei tests performed in 10 different laboratories was established. Data were available from over 4000 negative control rats from 10 laboratories. The mean frequency of micronucleated cells (MN)/1000 cells ranged from 0.44 to 2.22, a 5-fold range. Overall there were no major sex or strain differences in frequency, although there were some small but statistically significant differences within laboratories. There was appreciable variability between experiments compared with variability within experiments in some laboratories. No specific factor was identified which could explain this variability although it was noted that many different vehicles were used in the experiments. It is hoped that these data will help laboratories beginning studies with the rat micronucleus assay and those involved in the assessment of micronucleus assay results.

Human retinoic acid (RA) 4-hydroxylase (CYP26) is highly specific for all-trans-RA and can be induced through RA receptors in human breast and colon carcinoma cells.

We report on the isolation of a cytochrome P450 (CYP)-like retinoic acid (RA) 4-hydroxylase cDNA from T-47D human breast cancer cells that is identical to the recently cloned hCYP26, which is involved in the metabolic breakdown of RA. Northern analysis showed that this novel human CYP26 is induced within 1 h upon RA treatment in RA-sensitive T-47D breast carcinoma cells but not in RA-resistant MDA-MB-231 breast cancer cells and HCT 116 colon cancer cells. Stable introduction of different RA receptor (RAR) subtypes in HCT 116 cells showed that CYP26 expression is dependent on RARalpha and RARgamma and, to a lesser extent, on RARbeta and closely paralleled RA metabolism, suggesting that it represents the major RA 4-hydroxylase in these human cells. Furthermore, stable introduction of all three RAR subtypes in HCT 116 cells resulted in restored RA sensitivity as assayed by growth inhibition. Interestingly, CYP26 activity was efficiently inhibited by liarozole, an inhibitor of RA metabolism, leading to enhanced growth inhibition by RA. The RA-induced CYP26 was shown to be highly specific for the hydroxylation of all-trans-RA and did not recognize the 13-cis and 9-cis isomers. This substrate specificity is promising for finding retinoids that are not recognized by this enzyme and, therefore, could be more effective in growth inhibition of susceptible cancer cells.

Autoinduction of retinoic acid metabolism to polar derivatives with decreased biological activity in retinoic acid-sensitive, but not in retinoic acid-resistant human breast cancer cells.

Previous studies have shown that all-trans-retinoic acid (RA) inhibits in vitro proliferation of hormone-dependent human breast cancer cells but not the growth of hormone-independent cells. Here we report on RA metabolism in breast cancer cells as examined by high performance liquid chromatography analysis and found a correlation with sensitivity to growth inhibition by RA. RA-sensitive T-47D and MCF-7 cells exhibited high rate metabolism to polar metabolites, whereas RA-resistant MDA-MB-231 and MDA-MB-468 cells metabolized RA to a much lesser extent, and almost no polar metabolites could be detected. The high metabolic rate in RA-sensitive cells appears to be the result of autoinduction of RA metabolism, whereas RA-resistant cells showed no such induction of metabolism. We observed furthermore that transfection with retinoic acid receptor-alpha expression vectors in RA-resistant MDA-MB-231 cells resulted in increased RA metabolism and inhibition of cell proliferation. Metabolism of RA, however, seems not to be required to confer growth inhibition of human breast cancer cells. The biological activity of the polar metabolites formed in RA-sensitive cells was found to be equal or lower than that of RA, indicating that RA itself is the most active retinoid in these cells. Together our data suggest that RA-sensitive cells contain mechanisms to activate strongly the catabolism of RA probably to protect them from the continuous exposure to this active retinoid.

Novel progesterone target genes identified by an improved differential display technique suggest that progestin-induced growth inhibition of breast cancer cells coincides with enhancement of differentiation.

Progesterone is an important regulator of normal and malignant breast epithelial cells. In addition to stimulating development of normal mammary epithelium, it can be used to treat hormone-dependent breast tumors. However, the mechanism of growth inhibition by progestins is poorly understood, and only a limited number of progesterone target genes are known so far. We therefore decided to clone such target genes by means of differential display polymerase chain reaction. In this paper, we describe an improved differential display strategy that eliminates false positives, along with the identification of nine positive (TSC-22, CD-9, Na+/K+-ATPase alpha1, desmoplakin, CD-59, FKBP51, and three unknown genes) and one negative progesterone target genes (annexin-VI) from the mammary carcinoma cell line T47D, which is growth-inhibited by progestins. None of these genes have been reported before to be progesterone targets. Regulation of desmoplakin, CD-9, CD-59, Na+/K+-ATPase alpha1, and annexin-VI by the progestin suggests that progesterone induces T47D cells to differentiate. Three of these genes were repressed by estradiol and up-regulated by the progestin. Estradiol treatment of T47D cells also leads to formation of lamellipodia and delocalization of two cell adhesion proteins, E-cadherin and alpha-catenin. All these effects were reversed by the progestin. These data suggest that estradiol dedifferentiates T47D cells, while progestins have the opposite effect. This may be linked to the capacity of progestins to inhibit tumor growth.

[Retinoids: use in combating cancer]. / Retinoïden: toepassing bij de bestrijding van kanker.

Retinoids are a group of natural and synthetic analogues of vitamin A (retinol). The biological activity of retinoids is mediated by multiple nuclear receptors which bind to specific DNA sequences and regulate transcription of target genes. Retinoids affect cell growth and differentiation. Retinoids inhibit chemically induced carcinogenesis in experimental animals. Clinical application of retinoids is efficacious to different degrees in prevention and therapy of some malignancies in humans, such as promyelocytic leukaemia, notably in combination chemotherapy. Unfortunately, lack of response, recurrence, toxicity and resistance are observed. Worldwide clinical trials have been set up to evaluate the treatment of mammary carcinoma with retinoids. It appears important to determine, before starting treatment, whether oestrogen or retinyl receptors are present in the tumor tissue.

CYP2C7 expression in rat liver and hepatocytes: regulation by retinoids.

Rats deficient in vitamin A express low levels of P4502C7 mRNA in the liver. Administration of all-trans retinoic acid (at-RA) or growth hormone (GH) to deficient animals only partially restored the expression whereas the combined treatment returned the P4502C7 mRNA levels to that observed in normal rats. That a retinoid is the predominant inducer of P4502C7 at the cellular level is evident from studies performed with primary hepatocytes, but it became clear that GH is a prerequisite for the vitamin A effect in vivo. The at-RA induction of P4502C7 mRNA in primary rat hepatocytes was inhibited by ketoconazole, an inhibitor of P450 activity, and by cycloheximide, blocking ongoing protein synthesis. In contrast, the at-RA induction of RAR-beta2 mRNA was not affected by any of these compounds. This could indicate previously not recognized mechanisms of at-RA action. Interestingly, at-4-oxo-RA, an at-RA metabolite formed by a P450 catalyzed reaction, also induced P4502C7 mRNA. Induction of P4502C7 mRNA by the retinoic acid receptor (RAR) selective agonist TTNPB indicated that this pathway is preferred over the retinoid X receptor (RXR) pathway. In addition, analysis of RA metabolites in liver cell extracts revealed the formation of several as yet unidentified metabolites. The formation of some of these metabolites was inhibited by ketoconazole and they could therefore constitute potential inducers of CYP2C7. We suggest that metabolism of at-RA, possibly by a P450 enzyme, is an important step in the at-RA induction of P4502C7.

Retinoid metabolism and all-trans retinoic acid-induced growth inhibition in head and neck squamous cell carcinoma cell lines.

Retinoids can reverse potentially premalignant lesions and prevent second primary tumours in patients with head and neck squamous cell carcinoma (HNSCC). Furthermore, it has been reported that acquired resistance to all-trans retinoic acid (RA) in leukaemia is associated with decreased plasma peak levels, probably the result of enhanced retinoid metabolism. The aim of this study was to investigate the metabolism of retinoids and relate this to growth inhibition in HNSCC. Three HNSCC cell lines were selected on the basis of a large variation in the all-trans RA-induced growth inhibition. Cells were exposed to 9.5 nM (radioactive) for 4 and 24 h, and to 1 and 10 microM (nonradioactive) all-trans RA for 4, 24, 48 and 72 h, and medium and cells were analysed for retinoid metabolites. At all concentrations studied, the amount of growth inhibition was proportional to the extent at which all-trans-, 13- and 9-cis RA disappeared from the medium as well as from the cells. This turnover process coincided with the formation of a group of as yet unidentified polar retinoid metabolites. The level of mRNA of cellular RA-binding protein II (CRABP-II), involved in retinoid homeostasis, was inversely proportional to growth inhibition. These findings indicate that for HNSCC retinoid metabolism may be associated with growth inhibition.

Immunohistochemical analysis of retinoic acid receptor-alpha in human breast tumors: retinoic acid receptor-alpha expression correlates with proliferative activity.

Retinoids are known to prevent mammary carcinogenesis in rodents and inhibit the growth of human breast cancer cells in vitro. Previously we demonstrated that retinoid inhibition of proliferation of human breast cancer cell lines is largely mediated by retinoic acid receptor (RAR)-alpha. In this study we describe for the first time the histological distribution of RAR-alpha in 33 breast lesion specimens as determined by immunostaining with RAR-alpha antibody. Nuclear staining was observed in tumor tissue and normal portions of the breast samples. Connective tissue exhibited relative uniform staining, whereas a wide range of RAR-alpha expression was found in the epithelial tumor cells. RAR-alpha protein was expressed at significantly higher levels in tumors with greater proliferative activity as determined by immunostaining with Ki-67 antibody. This suggests that RAR-alpha expression may be altered with tumor progression. Although a positive correlation between RAR-alpha mRNA levels and estrogen receptor status of breast tumors has previously been documented, we did not find such a relationship at the protein level. As RAR-alpha plays a major role in retinoid-mediated growth inhibition of human breast cancer cell in vitro, our findings suggest that patients with highly proliferating tumors could be responsive to retinoid independently of their responsiveness to (anti)-estrogens.

Differential regulation of AP1 activity by retinoic acid in hormone-dependent and -independent breast cancer cells.

We have studied the role of the AP1 transcription factor in the progression of human breast carcinomas. This progression is characterized by a loss of dependence for proliferation on mitogenic hormones, and is also linked to loss of responsiveness to the growth inhibitor retinoic acid (RA). In the hormone-dependent breast tumor cell line MCF7 mitogenic stimulation was found to be linked to an enhancement of AP1 transcriptional activity, while growth inhibition by RA was parallelled by decreased AP1 activity. AP1 binding activity to its consensus DNA sequence was rapidly reduced in RA treated cells, in the absence of any noticeable change in expression of AP1 constituents. AP1 overexpression abrogated RA repression in MCF7 cells. In hormone-independent cell lines (BT20, Hs578T, MDA-MB231, MDA-MB468) autonomous proliferation was associated with an increased background AP1 activity. Interestingly, these cells are refractory to growth inhibition by RA, which can only be partly explained by underexpression of RA receptors. In these cells RA did not repress AP1 transactivation unless RA receptors were overexpressed by means of cotransfection with an expression vector. This suggests that the high background levels of AP1 activity in the autonomously growing cells are associated with prevention of RA inhibition of AP1 activity to occur. Therefore, increased AP1 activity may not only play a role in progression of breast tumors towards hormone-insensitivity but may also contribute to the RA resistance of such cells.

Retinoic acid resistance of estradiol-independent breast cancer cells coincides with diminished retinoic acid receptor function.

Retinoic acid (RA) strongly inhibits proliferation of the estrogen (E2)-dependent human breast cancer cell lines MCF7, T47D, and ZR75-1, but not the E2-independent and E2 receptor (ER)-negative lines MDA-MB231, MDA-MB468, BT20 and Hs578T. The specific sensitivity of the E2-dependent cell lines seems not to be caused by an inhibitory effect of RA on ER functioning since RA inhibited the proliferative response not only to E2 but also to insulin. Furthermore, endogenous RA receptors (RARs) hardly impaired transcriptional activation of an E2 responsive element-tk-CAT reporter construct. RAR alpha mRNA was highly expressed in the RA-responsive lines, but not in the unresponsive lines, except BT20. With the exception of Hs578T, also RAR beta mRNA expression was low in the unresponsive lines. While in the dependent lines and Hs578T RA activated RA responsive element-dependent transcriptional activity, this response was very low in MDA-MB231, MDA-MB468, and BT20, suggesting that the RA resistance of these latter three ER-negative lines is due to underexpression of functional RARs. Our results suggest that the loss of functional RARs may be a frequent event, leading to RA unresponsiveness of ER-negative breast cancer cells. This implies that both the steroid and retinoid receptor status of breast tumors may be used to predict a successful treatment with retinoids.

Retinoic acid receptor and retinoid X receptor expression in retinoic acid-resistant human tumor cell lines.

Retinoic acid (RA) has profound effects on cell proliferation and differentiation both in vitro and in vivo. Many human cell lines are known to be sensitive to the growth-inhibitory action of RA. We analyzed established human solid tumor-derived cell lines for their RA sensitivity. Growth inhibition by RA in monolayer was examined by [3H]thymidine incorporation and cell proliferation. Here we report that 11 widely used human cell lines were RA resistant. The majority are carcinoma derived (A-431, BT-20, C-41, ACHN, HCT116, 293, A549, and PA-1); two are sarcoma derived (Saos-2 and A673); and one is a melanoma cell line (A-375). Since nuclear retinoid receptors are implicated in the biological effects of RA, we examined the expression of retinoic acid receptors (RARs) RAR alpha, RAR beta, RAR gamma, and the retinoid X receptors (RXRs) RXR alpha, RXR beta, and RXR gamma in the RA-resistant cell lines by northern blotting and by RNase protection analysis for RAR beta. RAR alpha transcripts were constitutively expressed in all cell lines. By contrast, RAR beta was expressed in only seven RA-resistant cell lines (Saos-2, ACHN, 293, A549, A-375, A673, and PA-1), and its level was enhanced by RA in some cases. In most cell lines, RAR gamma expression was low and was not affected by RA. The RXR genes showed a very distinct expression pattern in the group of selected cell lines. In general, RXR alpha was the most abundantly expressed subtype, RXR beta was expressed at low levels, and RXR gamma could not be detected. In none of the RA-resistant cell lines was RXR expression modulated by RA. The results presented here indicate that the resistance of these human tumor cell lines to RA cannot be simply correlated with expression of RAR or RXR or both.