Comparative effects of sub-stimulating concentrations of non-human versus human Luteinizing Hormones (LH) or chorionic gonadotropins (CG) on adenylate cyclase activation by forskolin in MLTC cells.

We have compared various Luteinizing Hormone (LH) and Chorionic Gonadotropin (CG) preparations from non-human and human species in their ability to synergize with 10 µM forskolin (FSK) for cyclic AMP intracellular accumulation, in MLTC cells. LH from rat pituitary as well as various isoforms of pituitary ovine, bovine, porcine, equine and human LHs and equine and human CG were studied. In addition, recombinant human LH and CG were also compared with the natural human and non-human hormones. Sub-stimulating concentrations of all LHs and CGs (2-100 pM) were found to stimulate cyclic AMP accumulation in MLTC cells in the presence of an also non-stimulating FSK concentration (10 µM). Like rat LH, the most homologous available hormone for mouse MLTC cells, all non-human LHs and CG exhibit a strong potentiating effect on FSK response. The human, natural and recombinant hLH and hCG also do so but in addition, they were found to elicit a permissive effect on FSK stimulation. Indeed, when incubated alone with MLTC cells at non-stimulating concentrations (2-70 pM) hLH and hCG permit, after being removed, a dose-dependent cyclic AMP accumulation with 10 µM FSK. Our data show a clearcut difference between human LH and CG compared to their non-human counterparts on MLTC cells adenylate cyclase activity control. This points out the risk of using hCG as a reference ligand for LHR in studies using non-human cells.

Proliferative effect of histamine on MA-10 Leydig tumor cells mediated through HRH2 activation, transient elevation in cAMP production, and increased extracellular signal-regulated kinase phosphorylation levels.

Mast cells (MC) occur normally in the testis with a species-specific distribution, yet their precise role remains unclear. Testicular MC express histidine decarboxylase (HDC), the unique enzyme responsible for histamine (HA) generation. Evidence to date supports a role for HA as a local regulator of steroidogenesis via functional H1 and H2 receptor subtypes (HRH1 and HRH2, respectively) present in Leydig cells. Given that HA is a well-known modulator of physiological and pathological proliferation in many different cell types, we aimed in the present study to evaluate whether HA might contribute to the regulation of Leydig cell number as well as to the control of androgen production. Herein, we demonstrate, to our knowledge for the first time, that MA-10 Leydig tumor cells, but not normal immature Leydig cells (ILC), exhibit a proliferative response upon stimulation with HA that involves HRH2 activation, transient elevation of cAMP levels, and increased extracellular signal-regulated kinase (ERK) phosphorylation. Our results also reveal that MA-10 cells show significantly heightened HDC expression compared to normal ILC or whole-testicular lysate and that inhibition of HDC activity decreases MA-10 cell proliferation, suggesting a possible correlation between autocrine overproduction of HA and abnormally increased proliferation in Leydig cells. The facts that germ cells are also both source and target of HA and that multiple testicular cells are susceptible to HA action underline the importance of the present study, which we hope will serve as a first step for further research into regulation of non-MC-related HDC expression within the testis and its significance for testicular function.

Detection of aromatase and estrogen receptors (ERalpha, ERbeta1, ERbeta2) in human Leydig cell tumor.

A Leydig cell tumor is a rare neoplasm, deriving from the interstitial cells, whose pathogenesis has not been still defined. Leydig cells of normal adult testis are known as physiological targets for estrogens. However, some studies on transgenic rodents suggest a role of estrogens in the development of Leydig cell hyperplasia and Leydig cell tumor. Therefore, with the aim to evaluate a possible link between estrogens and testicular tumorigenesis, this study investigated the expression of aromatase and estrogen receptors (ERalpha, ERbeta(1), ERbeta(2)) in testes from two patients with Leydig cell tumor. A strong immunoreactivity for aromatase, ERbeta(1), and ERbeta(2), together with a detectable ERalpha immunostaining, was revealed in tumoral tissues. These findings were confirmed by western blot analysis of tumor extracts detecting a 55 kDa P450arom, a 67 kDa ERalpha band, a 59 kDa ERbeta(1) band, and a 53 kDa ERbeta(2) band. The pattern of ER expression in neoplastic cells appears different from that of control Leydig cells exhibiting only ERbeta(1) and ERbeta(2) isoforms. The authors hypothesize how the high estrogen production could play a role in the neoplastic transformation of Leydig cells, while the exclusive presence of ERalpha in tumoral cells could amplify estradiol-17beta signaling contributing to the tumor cell growth and progression.

Evidence for nitrite reductase activity in intact mouse Leydig tumor cells.

Nitric oxide (NO) supposedly derived via L-arginine-NO synthase (NOS) pathway has been implicated in inhibiting steroidogenesis by binding the heme moiety of steroidogenic enzymes. Previously, nitrite, and to a lesser extent nitrate ions inhibited steroidogenesis via NO by hitherto unknown reduction mechanism. Recently, a putative mammalian nitrite reductase activity ascribed to complex III of mitochondrial respiratory chain complexes (MRCC) has been reported, where MRCC inhibitors reduced NO production from nitrite variably. We thus studied the effects of MRCC inhibitors on testosterone production in mouse Leydig tumor cells (MLTC-1) without (basal) or with human chorionic gonadotropin (hCG) stimulation. In stimulated MLTC-1, MRCC inhibitors decreased testosterone production, order being: complex III (antimycin A and myxothiazol) > complex I (rotenone) > complex II (thenoyltrifluoroacetone), while cAMP production increased inversely. In unstimulated MLTC-1, MRCC inhibitors in same order, increased basal testosterone production, which correlated inversely with the percentage inhibition of NO production, with one exception; while antimycin A did not inhibit NO production in the nitrite reductase study mentioned above, it increased basal testosterone production in the present study. While MLTC-1 expressed mRNA for endothelial and neuronal, but not inducible NOS, various stimulators and inhibitors of L-arginine-NOS pathway had no effect on basal testosterone production in MLTC-1 or fresh Balb/c Leydig cells. Moreover, hCG increased nitrate uptake into MLTC-1, which suggests the gonadotropin aids nitrite and nitrate ions in their steroidogenesis inhibitory activity. In conclusion, this study supports the existence of a surrogate mammalian nitrite reductase and the dormancy of L-arginine-NOS pathway in MLTC-1.

DNA synthesis and DNA polymerase activity in Leydig cells of diethylstilbestrol-stimulated mouse testes.

Using a modification of the collagenase dispersion method of Dufau et al., we examined changes in DNA synthesis produced by estrogens in the interstitial cells of mice that develop malignant Leydig cell tumors after prolonged estrogen administration. Previous work in cryptorchid mice indicated that during continuous estrogen administration [3H]thymidine incorporation into DNA rises to a maximum in 3 to 4 days and then falls to approximately base levels within 2 to 3 weeks. This was confirmed both in Leydig cell concentrates of estrogen-treated mice after either injection with [3H]thymidine or incubation with [3H]thymidine in vitro. This DNA synthesis was blocked by hydroxyurea. DNA synthesis in cells of estrogen-treated BALB/c mice of the Huseby substrain, which have a high incidence of Leydig cell tumors, was 5 to 11 times that in untreated controls. Cells from estrogen-treated C3H/Bi mice, which have a low incidence of Leydig cell tumors, showed only a 2- to 3-fold increase. In the Huseby substrain the rise of DNA synthesis is a peak and subsequent recession were paralleled by a rise and fall in DNA polymerase alpha activity. DNA polymerase beta did not show this variation. In C3H/Bi mice, neither polymerase showed significant change. The evidence suggests that the early estrogen-stimulated DNA synthesis is probably replicative and is associated with increased DNA polymerase alpha activity.

Aneuploidy of human testicular germ cell tumors is associated with amplification of centrosomes.

Testicular germ cell tumors occur in three age groups. Seminomas and nonseminomas of adults, including mature teratomas, and the precursor carcinoma in situ (CIS) are aneuploid. This also holds true for yolk sac tumors of newborn and infants, while the mature teratomas of this age are diploid. In contrast, spermatocytic seminomas occurring in the elderly contain both diploid and polyploid cells. Aneuploidy has been associated with centrosome aberrations, sometimes related to overexpression of STK15. Aneuploidy of non-neoplastic germ cells has been demonstrated in the context of male infertility, a risk factor for the development of seminoma/nonseminoma. We investigated aneuploidy, centrosome aberrations and the role of STK15 in different types of testicular germ cell tumors as well as in normal and disturbed spermatogenesis. The aneuploid seminomas and nonseminomas tumors (including CIS) showed increased numbers of centrosomes, without STK15 amplification or overexpression. Four out of six infantile teratomas had normal centrosomes, the remaining two and an infantile yolk sac tumor showed a heterogeneous pattern of cells with normal or amplified centrosomes. Spermatocytic seminomas had two, four or eight centrosomes. Germ cells in seminiferous tubules with disturbed spermatogenesis shared both aneuploidy and centrosome abnormalities with seminomas/nonseminomas and showed a more intense STK15 staining than those with normal spermatogenesis and CIS. Therefore, aneuploidy of testicular germ cell tumors is associated with amplified centrosomes probably unrelated to STK15.

Characterization of protein tyrosine kinase activity in murine Leydig tumor cells.

The activity of protein tyrosine kinase (EC 2.7.1.37) was characterized from Leydig tumor cells (M5480A) using the synthetic peptide NH2-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly-COOH as a substrate. Relatively high tyrosine-specific protein kinase activity (about 135 pmol/mg protein per min) was detected in a particulate fraction (30 000 X g pellet) and was found to be linear as a function of time and protein concentration. The enzymic activity in the particulate fraction was stimulated 1.4-fold by 0.02% Nonidet P-40 as judged by 32PO4 incorporated into the peptide. Phosphorylation of endogenous proteins in M5480A particulate fractions with [gamma-32P]ATP resulted in several alkali-resistant radiolabeled bands in polyacrylamide gels in the presence of sodium dodecyl sulfate. Included in this group was a major radiolabeled doublet with an apparent molecular-weight in the range of 50 000-54 000. Phosphoamino acid analysis of hydrolysates of these eluted proteins indicated the presence of phosphotyrosine. Several alkali-resistant radio-labeled bands, including a major doublet with an apparent molecular-weight of 32 000, were also detected after culturing M5480A cells in the presence of 32PO4. These studies demonstrate the presence of high levels of protein tyrosine kinase activity in Leydig tumor cells and of endogenous protein substrates for this enzyme activity.

The gonadotropin-regulated long-chain acyl CoA synthetase gene: a novel downstream Sp1/Sp3 binding element critical for transcriptional promoter activity.

The 79 kD gonadotropin-regulated testicular long chain acyl-CoA synthetase gene (GR-LACS) is a hormone-regulated member of the acyl-CoA synthetase family that is expressed abundantly in Leydig cells and to a lesser extent in germinal cells of the adult testis. GR-LACS possesses an ATP/AMP binding domain and the fatty acyl-CoA synthetase (FACS) signature motif. To gain insights into the transcriptional regulation of GR-LACS in gonadal cells, we determined the genomic organization of the gene, including the upstream flanking sequences. The mouse GR-LACS gene spans over at least 45 kb and the coding region is encoded by exons 1-14. All exon-intron junction sites correspond to the consensus splice sequence GT-AG. Exon 7 and 11 comprise the conserved ATP/AMP binding domain and the FACS signature motif, respectively. Primer extension and S1 nuclease analyses demonstrated four transcriptional start sites located at -266/-216 bp 5' to the ATG codon. The minimal promoter domain resides within -254/-217 bp 5' to ATG codon, and upstream sequences to -404 bp (-1035/-405 bp) contribute to the inhibition of transcription in the expressing mouse Leydig tumor cells. Removal of -217/-1 bp, containing a 23 nt GC rich sequence (-112/-90) with an Sp1/Sp3 binding element, within the 1st exon of this TATA-less promoter, significantly reduced GR-LACS gene transcription. Transcriptional activity was abolished by a 2 nt mutation of this element. Thus, functional analyses of this promoter domain indicate that transcription of GR-LACS gene requires an Sp1/Sp3 binding element downstream of the transcriptional start sites which is essential for basal promoter activity.

Inhibition of mTOR suppresses experimental liver tumours.

Sirolimus, and its antiproliferative capacity, was studied in vivo in three different syngenic rat tumours in the liver. Sirolimus is an inhibitor of the cytosolic mTOR-kinase, associated with the phosphoinositide-3-kinase/Akt pathway. After one week of daily sirolimus treatment, initiated on the day of tumour-cell inoculation, a dose-response relationship was shown at doses between 0.01 mg/kg/day and 1 mg/kg/day, decreasing tumour weight from 0.5+/-0.1 g in control rats (n=9) to 0.09+/-0.04 g for sirolimus 1 mg/kg (n=9). Treating established liver adenocarcinoma (n=15), sirolimus halved the tumour weight (1.4+/-0.2 g vs 0.7+/-0.1 g, p=0.005). Trough concentration in blood was 6.4+/-0.2 ng/ml after five days of daily treatment with 1 mg/kg sirolimus intraperitoneally. At this dose, there was no decrease in food consumption or rat weight, but decrease in weight of spleen, and increase in weight of liver (p<0.01). The three tumours studied, an nitrosoguanidin-induced adenocarcinoma, a Leydig cell sarcoma and a hepatoma, all responded, establishing sirolimus as a promising anticancer drug.

Estradiol acts as a competitive inhibitor of the 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase enzyme of cultured Leydig tumor cells.

To study the local regulatory mechanisms involving steroid hormones in steroidogenic cells, the effect of estradiol on steroidogenesis was investigated using MA-10 Leydig tumor cells. Estradiol inhibited progesterone biosynthesis in MA-10 cells in a dose-dependent manner. Inhibition of progesterone biosynthesis by estradiol was associated with a concomitant accumulation of pregnenolone in the incubation medium. Estradiol inhibited the activity of 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase by a chemical mechanism which is not mediated through the cellular estrogen receptor. Thus, the estrogen receptor agonist diethylstilbestrol did not inhibit this enzyme activity, nor could this agent block the effect of estradiol on the enzyme. Furthermore, estradiol inhibited enzyme activity in isolated microsomes which do not contain estradiol receptor protein. Kinetic analysis of the inhibitory effect of estradiol on 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase revealed that this steroid hormone functions as a competitive inhibitor of the enzyme, with an average apparent Ki of 1.8 microM.

Gonadotropin-mediated desensitization in a murine Leydig tumor cell line does not alter the regulatory and catalytic components of adenylate cyclase.

MLTC-1 cells, derived from a murine Leydig tumor, contain a gonadotropin-responsive adenylate cyclase that became desensitized to hCG. Prior exposure to hCG reduced the ability of MLTC-1 cells to accumulate cAMP by approximately 50%, but caused only a small reduction in hCG receptor number. Membranes isolated from desensitized cells showed a similar reduction in hCG-stimulated adenylate cyclase activity. Desensitization was time, temperature, and dose dependent. Elevating intracellular cAMP levels by incubating the cells with (Bu)2cAMP or cholera toxin failed to cause desensitization. Desensitization did not depend on protein synthesis. Desensitization caused no change in the dose response of adenylate cyclase to hCG or GTP. hCG receptor affinity for hCG was not affected by desensitization or guanine nucleotides. The stimulatory regulatory component of adenylate cyclase (Ns) from MLTC-1 cells was used to reconstitute S49 cyc- membranes, which lack Ns. Ns from control and desensitized MLTC-1 cells were equally effective in reconstitution of the beta-adrenergic-sensitive adenylate cyclase of cyc-. beta-Adrenergic receptors from cyc- membranes were also transferred to MLTC-1 membranes by fusion with polyethylene glycol to produce a beta-adrenergic-responsive adenylate cyclase. Isoproterenol-stimulated activity was similar, regardless of whether membranes from control or desensitized MLTC-1 cells were used. We conclude that neither Ns nor the catalytic subunit of the adenylate cyclase in MLTC-1 cells is the site of lesion in desensitization. Most likely, the hCG receptor itself may be affected when MLTC-1 cells are desensitized by hCG.

The possible roles of membrane organization in the activity of androgen biosynthetic enzymes associated with normal or tumorous mouse Leydig cell microsomes.

The enzymes involved in conversion of pregnenolone to testosterone in Leydig cell tumors showed a wide distribution among smooth endoplasmic reticulum (SER), rough endoplasmic reticulum (RER), and cytosol, while these enzymatic activities in normal testes were associated primarily with smooth endoplasmic reticulum. Progesterone, used as a substrate in the presence of an NADPH-generating system, was metabolized to androstenedione and finally to testosterone by microsomes from some strains of tumor which did not form testosterone from exogenous labeled androstenedione. Treatment of microsomal membranes from normal testes with 0.1 M Ca++ and Mg++ caused a marked decrease in 17 beta-dehydrogenase activity, measured as conversion of exogenous [3H]androstenedione to [3H]-testosterone, without serious effects on activities of 3 beta-ol-dehydrogenase or 17 alpha-hydroxylase. Studies of initial velocity kinetics showed that treatment with magnesium ion resulted in a marked reduction in affinity of androstenedione for 17 beta-dehydrogenase while the maximum velocity was the same as in untreated microsomes. Also, experiments using [14C]progesterone and [3H]androstenedione simultaneously as substrates demonstrated that treatment with Mg++ ion made it more difficult for exogenous [3H]androstenedione to reach the active site of 17 beta-ol-dehydrogenase than [14C]androstenedione formed in the microsomal membrane from [14C]progesterone. Microsomal proteins were more easily solubilized and 3 beta-ol-dehydrogenase was more severely influenced by Mg++ ion in tumor membranes than in normal microsomes.

Epidermal growth factor desensitizes the gonadotropin-responsive adenylyl cyclase in membranes isolated from MA-10 Leydig tumor cells and luteinized rat ovaries.

In a previous publication we showed that addition of mouse epidermal growth factor (mEGF) to MA-10 Leydig tumor cells or rat luteal cells leads to an attenuation of the elevated rate of cAMP accumulation provoked by subsequent addition of hCG. Those studies also suggested that this was due to a decrease in the hCG-activated adenylyl cyclase activity, but formal proof of this hypothesis was not presented. The experiments presented herein were conducted to investigate the mechanisms responsible for this phenomenon and show that our initial suggestion was correct, because we can show that mEGF attenuates the hCG-activated adenylyl cyclase in membranes from MA-10 cells or luteinized rat ovaries. More importantly, however, the establishment of a cell-free system in which mEGF attenuates adenylyl cyclase allows us to conduct detailed investigations on the molecular mechanisms that underly this phenomenon. Using this system we have been able to show that the ability of mEGF to attenuate adenylyl cyclase activity 1) is specific for the hCG-activated enzyme, since it cannot be detected when the enzyme is activated with other ligands, such as vasoactive intestinal peptide or isoproterenol; 2) does not appear to be due to a reduction in the activity of the catalytic subunit of adenylyl cyclase or the stimulatory GTP-binding protein (Gs), because mEGF does not affect the stimulation of adenylyl cyclase by other effectors, and it has little or no effect on the ability of Gs to restore isoproterenol sensitivity to the adenylyl cyclase of S49 cyc- membranes; and 3) optimal expression of the mEGF effect can only be obtained in the presence of ATP or GTP.

Aromatase activity in a rat Leydig cell tumor.

Male Wistar-Furth rats bearing the transplantable LTW(m) Leydig cell tumor have elevated serum estradiol (E2) concentrations. We measured the ability of these tumors to aromatize testosterone (T) to E2 by two methods. First, tumor minces were incubated with [7-3H]T, and the resultant [3H]E2 and [3H]estrone were purified and measured. In addition, tumor cell cultures were incubated with [1 beta-3H]T, and the resultant [3H]H2O was determined as a measure of aromatization. Tumor minces aromatized more actively than normal rat testicular tissue (3.30 +/- 0.15% of the T added was converted to E2 by the tumor vs. 0.30 +/- 0.25% by normal testis). Most of the aromatizing actitivity was localized to the microsomes. Using cell cultures the maximum velocity was 6.1 pmol/h X 5 X 10(5) cells, and the Km was 98 nM. In neither minces nor cell cultures were we able to show stimulation of aromatization with hCG, (Bu)2cAMP, or phorbol esters, although we could show stimulation by these agents in normal testicular cells. We were unable to inhibit the aromatase activity with human beta-endorphin or stimulate it with naloxone. However, we were able to inhibit the aromatase activity with 4-hydroxy-4-androstene-3,17-dione. We conclude that the LTW(m) rat Leydig cell tumor has an active autonomous aromatase system that is not responsive to compounds affecting the adenylate cyclase-cAMP system. It can be inhibited by 4-hydroxy-4-androstene-3,17-dione, a competitive-suicide inhibitor of the aromatase enzyme(s).

Induction of renin activity by gonadotropic hormones in cultured Leydig tumor cells.

The hormonal regulation of renin activity in cloned and cultured Leydig tumor cells (designated MA-10) was examined. The treatment of Leydig cell cultures with bovine LH (bLH), hCG, or with (Bu)2cAMP elicited a dose- and time-dependent induction of renin activity and a concomitant increase in steroid biosynthesis. The optimum concentration of hCG was 25 ng/ml, which caused an average 25-fold increase in renin activity compared to the control value. bLH action was optimum at 75-100 ng/ml and induced an approximately 35-fold increase in renin activity. The maximum inducible level of renin activity was attained after 8-9 h of hormone treatments. The addition of progesterone (the major steroid product of the MA-10 cells) did not induce a significant increase in renin activity. Treatment of MA-10 cells with epidermal growth factor also failed to produce any increase in renin activity. The optimum concentration of (Bu)2cAMP was 800 microM for the induction of renin activity and caused an approximately 40-fold increase compared to the control value. Renin activity induced by bLH, hCG, or (Bu)2cAMP was completely inhibited by mouse anti-renin antibody, indicating the specific nature of renin. Upon withdrawal of (Bu)2cAMP from the culture medium, renin activity gradually declined to the control level, and with retreatment of these cultures with (Bu)2cAMP, a newly induced state of enzyme activity was resumed. Indirectly, the role of new protein and RNA synthesis was examined during hormonal regulation of renin induction using protein and RNA synthesis inhibitors such as cycloheximide, puromycin, actinomycin D, or rifampicin. Both protein and RNA synthesis inhibitors blocked the induction of renin activity in the presence of all three inducing agents, bLH, hCG, or (Bu)2cAMP. The results provide evidence that the induction of renin activity is modulated by bLH, hCG, or (Bu)2cAMP and represent the de novo synthesis of enzyme molecules.

Glucocorticoid and cyclic adenosine 3'5'-monophosphate-mediated induction of cholesterol side-chain cleavage cytochrome P450 (P450scc) in MA-10 tumor Leydig cells. Increases in mRNA are cycloheximide sensitive.

The regulation of cholesterol side-chain cleavage enzyme (P450scc) was investigated in MA-10 tumor Leydig cells. We recently demonstrated that the constitutive and cAMP-stimulated expression of P450scc in normal mouse Leydig cells is negatively regulated by glucocorticoids. We now report that glucocorticoids have the opposite effect in MA-10 cells causing a 1.7-fold increase in the rate of P450scc synthesis and a 2.1-fold increase in the amount of P450scc mRNA. Treatment of MA-10 cells with 10 microM 8-bromo-cAMP (8-Br-cAMP) (cAMP) resulted in a 1.7-fold increase in P450scc synthesis and a 3-fold increase in P450scc mRNA. Combined treatment with dexamethasone and cAMP resulted in additive increases in synthesis (2.8-fold) and mRNA (5.3-fold). Increases in de novo synthesis and mRNA levels were reflected by modest increases in the amount of immunoreactive P450scc enzyme protein. Dexamethasone-mediated stimulation in synthesis and accumulation of P450scc mRNA were blocked by the antiglucocorticoid RU-486. Cycloheximide blocked both cAMP- and dexamethasone-induced increases but had no effect on constitutive levels of P450scc mRNA. Treatment of MA-10 cells with 10 microM 8-Br-cAMP had no effect on cell morphology and stimulated progesterone accumulation to a minor degree. Treatment of MA-10 cells with 1 mM 8-Br-cAMP resulted in cell rounding and loss of cells from culture dishes. The results of this study demonstrate that: 1) dexamethasone increases P450scc de novo synthesis and mRNA levels in MA-10 tumor Leydig cells, opposite to the effect in normal Leydig cells; 2) dexamethasone- and cAMP-stimulated increases occur via distinct mechanisms; 3) and synthesis of protein factor(s) is required to mediate the action of both dexamethasone and cAMP.

Synthetic and partially-purified adenylate cyclase-stimulating proteins from tumors associated with humoral hypercalcemia of malignancy inhibit phosphate transport in a PTH-responsive renal cell line.

Hypophosphatemia and hyperphosphaturia characteristically occur in patients with humoral hypercalcemia of malignancy (HHM). To determine if a tumor product causes these abnormalities in phosphate metabolism, rather than, for example, hypercalcemia, we investigated the effect of partially-purified adenylate cyclase-stimulating activity (ACSA) from human and animal HHM-associated tumors on sodium-dependent phosphate transport (Na PiT) in a PTH-responsive renal epithelial cell line. Thirty minute exposure to 7 X 10(-10) MbPTH (1-34) equivalents of ACSA from the human and animal tumors, reduced NaPiT by 20% and 14%, respectively. We also recently isolated an adenylate cyclase-stimulating protein (hACSP) from two human tumors associated with HHM and identified a cDNA clone for this protein which encodes a 141 amino-acid peptide. Based on the deduced amino-acid sequence, we synthesized tyr36 (1-36) hACSP. This synthetic peptide induced a 22% decrease in the initial rate of NaPiT by the epithelial monolayer. Its inhibitory activity was roughly equipotent to that of bPTH (1-34). We conclude that the ACSP derived from HHM-associated tumors decreases phosphate transport in renal epithelial cells. This peptide appears to play a key role in mediating the changes in phosphate metabolism in this syndrome.

Cortisol production by testicular tumors in a patient with congenital adrenal hyperplasia (21-hydroxylase deficiency).

Bilateral testicular tumors are known to occur in congenital adrenal hyperplasia, but their steroidogenic properties are not well studied. We have recently demonstrated steroid 11 beta-hydroxylase activity in these tumors from a 27-yr-old patient with documented salt-losing congenital hyperplasia (21-hydroxylase deficiency). Spermatic venous blood obtained at operation contained 3.6-5 ng/ml cortisol, as measured by specific RIA after Sephadex LH-20 chromatography. In contrast, the peripheral venous cortisol level was 0.64 ng/ml. The presence of 11 beta-hydroxylase activity was demonstrated by the in vitro conversion of [3H]deoxycorticosterone to [3H]corticosterone in tumor tissue. The conversion ratio of deoxycorticosterone to corticosterone was 4.3% and 7.07%/mg tissue in the tumor tissue, whereas in normal testicular tissue, it was less than 1%. The radiochemical identity of corticosterone was confirmed by rechromatography on Sephadex LH-20, coelution with added [14C]corticosterone, and a constant 3H to 14C ratio after acetylation and thin layer chromatography. The tumor cells grown in primary culture produced steroids and responded to ACTH and hCG. Electron microscopy of the tumor revealed a large number of mitochondria containing electron dense granules. Light microscopy was compatible with Leydig cell tumor. In conclusion, testicular tumors in association with congenital adrenal hyperplasia have morphological features of Leydig cells but have the capability of 11 beta-hydroxylation and cortisol production, properties which are unique to adrenocortical tissue. These findings suggest that they originate from pluripotential cells in the testicles. Regression of these tumors has been reported with optimal biochemical control of the disease.

Stimulation of cholesterol side-chain cleavage enzyme activity by cAMP and hCG in MA-10 Leydig tumor cells.

Using a cloned Leydig tumor cell line (designated MA-10), we have studied the activity of cholesterol side-chain (CSCC) enzyme, the rate-determining step in steroidogenesis, in mitochondria isolated from cells pretreated either with human chorionic gonadotropin (hCG) or dibutyryl cyclic adenosine monophosphate (dbcAMP). Results showed a slight but significant increase in CSCC activity with treatment by cAMP (25% increase) and hCG (60% increase), as compared to mitochondria isolated from nontreated control cells. However, this stimulation of CSCC activity appears to be of limited significance when compared to the approximately 1000-fold or greater increase observed in progesterone production in the presence of hCG or dbcAMP. On the other hand, unstimulated MA-10 cells or isolated mitochondria efficiently converted 25-hydroxycholesterol and 22R-hydroxycholesterol into progesterone, and this conversion was not affected by cycloheximide. The addition of cholesterol to intact cells or to isolated mitochondria did not affect progesterone production. Our observations clearly indicate that given the proper hydroxy substrates (22R-hydroxycholesterol or 25-hydroxycholesterol), MA-10 Leydig cells are able to convert them into progesterone without any stimulation by steroidogenic stimuli, i.e. cAMP or hCG. Since MA-10 Leydig cells can efficiently convert 22R-hydroxycholesterol--an intermediate in CSCC reaction--into progesterone, these results suggest that the key regulatory step in the mechanism of trophic hormone-stimulated steroid production is the first hydroxylation step of the 3 sequential monooxygenation reactions involved in the conversion of cholesterol to pregnenolone.

Effects of troglitazone on intracellular cholesterol distribution and cholesterol-dependent cell functions in MA-10 Leydig tumor cells.

Troglitazone treatment of MA-10 Leydig tumor cells resulted in cellular cholesteryl esters decreasing and cell free cholesterol increasing. This was not an effect unique to this chemical entity; rosiglitazone and pioglitazone caused these changes also. The excess free cholesterol was recovered largely in the cholesterol oxidase susceptible, plasma membrane cholesterol pool. This effect of troglitazone probably is not mediated by activation of peroxisome proliferator activated receptors since it immediately reversed with washing and did not occur at all in cells treated with the peroxisome proliferator activated receptor agonist, 15-deoxy Delta 12,14 prostaglandin J-2. Plasma membrane cholesterol esterification was inhibited by troglitazone in a dose-dependent manner. Plasma membrane cholesterol esterification was inhibited half-maximally by 14 microM troglitazone and by more than 90% by 40 microM troglitazone. This effect was not unique for MA-10 cells. Similar results were found using fibroblasts. Troglitazone was not simply inhibiting internalization of plasma membrane cholesterol. Dibutyryl-cAMP stimulation of troglitazone-treated cells resulted in more progesterone synthesis than in stimulated control cells; moreover, radioactive plasma membrane cholesterol was readily converted into progesterone in troglitazone-treated cells. Studies of LDL uptake in troglitazone-treated cells indicated that intracellular membranes were cholesterol replete. Troglitazone inhibited plasma membrane cholesterol esterification with kinetics similar to 58-035, a known inhibitor of the acyl coenzyme A: cholesterol acyltranserase (ACAT) enzyme. It is not likely an ACAT inhibitor since troglitazone did not block incorporation of exogenous free fatty acids into cholesteryl esters. Thus, it appears that troglitazone prevented presentation of free fatty acid to the ACAT enzyme.