Identification and differential expression of human collagenase-3 mRNA species derived from internal deletion, alternative splicing, and different polyadenylation and transcription initiation sites.

OBJECTIVE: Collagenase-3 is a metalloprotease that plays a role in tissue remodeling and pathological processes including arthritis. The human gene is transcribed into major (3.0 and 2.5 kb) and minor (2.2/2.0 kb) transcripts, as seen in Northern blot assays. We investigated the possibility that other transcripts, not detectable by Northern blot, were synthesized as either coding or regulatory RNAs that would modulate collagenase-3 expression and function/activity. DESIGN: We screened a cDNA library and total RNA from human chondrocytes by plaque hybridization and RT-PCR, and expressed the transcripts in a cellular environment. The levels of expression of each transcript in normal and osteoarthritic joint cells and cartilage were monitored by RT-PCR. RESULTS: We identified five different collagenase-3 RNA species derived from alternative polyadenylation sites (COL3-APS), internal deletion (COL3-DEL), alternative splicing (COL3-9B/COL3-9B-2), and different transcription initiation site (COL3-ATS and COL3-ATS-INT). Each transcript could be translated in a cellular environment. Interestingly, the proteins translated from the COL3-DEL and COL3-9B-2 transcripts had a modified hemopexin-like domain, suggesting altered collagenolytic activities. The transcript types COL3-APS, COL3-9B-2, and COL3-ATS were up-regulated in the osteoarthritic samples and expressed in the chondrosarcoma cell line SW1353. CONCLUSION: Our data show that the human collagenase-3 gene is subjected to different levels of regulation and constitutes a more complex system than was originally thought.

Hormonal regulation of N-cadherin mRNA levels in rat granulosa cells.

We have examined the ability of hormones to modulate the steady-state levels of N-cadherin mRNA transcripts in aggregated and dispersed rat granulosa cell populations. Estradiol and follicle-stimulating hormone (FSH) had no effect on the levels of N-cadherin mRNA transcripts in aggregated granulosa cells. In contrast, these two hormones stimulated N-cadherin mRNA levels in dispersed granulosa cells. This is the first report that estradiol and FSH are capable of regulating N-cadherin mRNA levels. The results also suggest that the N-cadherin mRNA levels in dispersed and aggregated granulosa cells are regulated by different mechanisms.

Inhibitory effects of medroxyprogesterone acetate (MPA) and the pure antiestrogen EM-219 on estrone (E1)-stimulated growth of dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma in the rat.

Estrogens are well known to play a predominant role in promoting the growth of DMBA-induced mammary tumors in the rat. Estrone (E1), a steroid having weak estrogenic activity, is one of most important estrogens in post-menopausal women, where it is converted into the potent estrogen estradiol (E2) by 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) in many peripheral tissues, including the mammary gland. In this report, we have studied the effect of a new antiestrogen (EM-219) (N-butyl, N-methyl-11-(3', 17'beta-dihydroxy-17'alpha-ethinyl-estra-1'3'5'(10'), 14'-tetraen-7'alpha-yl) undecanamide) on E1-stimulated growth of DMBA-induced mammary tumors and compared its effect with that of medroxyprogesterone acetate (MPA) alone or in combination. After 18 days, ovariectomy (OVX) reduced total tumor area to 29.6 +/- 7.1% of the original size, while E1 (1.0 microgram, twice daily) caused a 139 +/- 21% increase in tumor size in OVX animals. MPA (1.5 mg, twice daily) partially reversed the stimulatory effect of E1 to 66.0 +/- 9.0%, while the antiestrogen EM-219 (40 micrograms, twice daily) decreased tumor size to 70.0 +/- 10%. Combination of these two compounds led to a further inhibition of tumor size to 30.7 +/- 7.4% of the value found in OVX animals treated with E1. Tumor E2 levels decreased from 1688 +/- 155 pmoles/kg tissue in OVX animals receiving E1 to 709 +/- 92, 1347 +/- 98, and 184 +/- 11 pmoles/kg tissue in MPA-, EM-219-, and MPA+EM-219-treated OVX-E1 animals, respectively. Treatment of OVX animals with E1 increased by 69% the reductive activity of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) while MPA abolished completely this effect of E1. In the oxidative direction, treatment with E1, E1 + MPA, or E1 + EM-219 had minimal or no significant effect on the activity of 17 beta-HSD (vs OVX), while the combined treatment with MPA+EM-219 induced a 2-fold increase in 17 beta-HSD activity, thus leading to an increased conversion of E2 into E1. The present data show that combination of the pure antiestrogen EM-219 with MPA exerts a greater reduction in DMBA-induced mammary tumor growth and intratumoral E2 levels stimulated by E1 than either compound used alone. This interactive effect of the antiestrogen and MPA could at least partially be related to the increased inactivation of E2 into E1.(ABSTRACT TRUNCATED AT 400 WORDS)

Additive inhibitory effects of an androgen and the antiestrogen EM-170 on estradiol-stimulated growth of human ZR-75-1 breast tumors in athymic mice.

The effects of the androgen dihydrotestosterone (DHT) and of the androgenic steroid medroxyprogesterone acetate were studied on the growth of human ZR-75-1 breast carcinoma in athymic mice. The possibility of additive inhibitory effects of DHT and the new steroidal antiestrogen N-n-butyl, N-methyl-11-[16' alpha-chloro-3',17' alpha-dihydroxyestra-1',3',5'(10')trien-7' alpha-yl]undecanamide (EM-170) was also investigated on tumor growth. Removal of the high dose 17 beta-estradiol (E2) implants used to optimally stimulate initial ZR-75-1 tumor development in ovariectomized mice led to a progressive decrease in tumor area to 50.2 +/- 8% (SEM) of original tumor size 40 days after E2 deprivation. Additional treatment with the androgen DHT led to a more rapid fall in tumor volume, which already reached 57% of pretreatment values at 11 days. Whereas physiological implants of E2 led to a progressive increase in tumor size to about 180% above original size after 40 days, physiological plasma levels (205 +/- 37.2 pg/ml or approximately 0.67 nM) of DHT completely reversed the stimulatory effect of E2. Similar inhibitory effects on E2-stimulated tumor growth were achieved with the synthetic androgenic steroid medroxyprogesterone acetate. When the steroidal antiestrogen EM-170 at the dose of 30 micrograms/day was used simultaneously with DHT, tumor area was further reduced from 99.0 +/- 9.5% (DHT alone) to 58.8 +/- 18% when both DHT and EM-170 were administered together for 40 days compared with 169 +/- 22.2% in control E2-stimulated animals. The present data show that the androgen DHT as well as medroxy-progesterone acetate are potent inhibitors of E2-stimulated human ZR-75-1 breast cancer cell growth in vivo. Moreover, the inhibitory effect of DHT can be further increased by addition of the antiestrogen EM-170, thus suggesting the interest of combining these 2 classes of compounds acting, at least partially, through different mechanisms, in order to improve breast cancer therapy in women.