Biomedical subjects

M R Sairam

Publications and source records attributed to M R Sairam.

At least 19 recordsLinked to original sources

PubMed1999

Structural features and expression of an alternatively spliced growth factor type I receptor for follitropin signaling in the developing ovary.

The pleiotropic actions of pituitary follitropin (FSH), regulate the expression of many cell cycle genes controlling ovarian follicular development and differentiation. In this study we asked the question whether different receptor motifs are created by the alternative splicing of the single large 80-100 Kb receptor gene. A 1.2 Kb transcript identified from a cDNA library of hormone primed (immature) sheep ovaries, codes for a putative protein lacking the seven transmembrane segment. The receptor of 259 amino acids designated FSH-R3 is derived from a transcript comprising the first eight exons of the Gs coupled larger FSH receptor (R1) spliced to another DNA segment. This event produces a different carboxyl terminus at the junction creating a novel receptor motif with a single membrane spanning domain, assigning it to the growth factor type I receptor family. In transfected cells the expressed receptor localizes on the cell surface and specific antibodies directed against the unique C-terminal portion (residues 242-259) of FSH-R3 demonstrate the presence of the receptor protein in solubilized ovarian and testicular membrane preparations. FSH binding to the transfected cells induced [Ca2+]i identifying coupling of the R3 receptor to calcium signaling pathways. Thus, a growth factor type I receptor for FSH may be implicated in the growth promoting actions of FSH in the ovary. This is the first documentation of alternative splicing of a G protein coupled receptor gene creating a different signaling motif for cellular signaling.

Alternative Splicing↗

PubMed1999

Hormonal regulation of natriuretic peptide system during induced ovarian follicular development in the rat.

All components of the natriuretic peptide (NP) system have been found in the ovary. The purpose of this study was to determine the hormonal regulation of the NP system during follicular growth and ovulation induced by gonadotropins eCG and hCG. Ovarian membrane binding, before and after treatment, revealed the presence of guanylyl cyclase-type receptors exclusively. Equine CG treatment increased Bmax from 225 +/- 50 fmol/mg protein in control animals to 354 +/- 51 fmol/mg protein, and additional hCG treatment increased it further to 492 +/- 130 fmol/mg protein (p < 0.05), without changing receptor affinity. The increased binding was consistent with increased ability of atrial natriuretic peptide (ANP) to activate guanylyl cyclase in the ovarian cells obtained from hormone-treated animals. In confirmation, autoradiography of 125I-tyroCNP and 125I-ANP binding to the rat ovary showed that both guanylyl cyclase GC-A and GC-B receptor subtypes are localized to the granulosa cells of antral follicles. Quantitative analysis of GC-A and GC-B receptors by reverse transcription-polymerase chain reaction showed that the expression level of both receptors started to increase at 2 h and reached maximal levels at 6 h following eCG treatment. Increased levels of GC-B mRNA were also observed 12 h after eCG injection. At 24 and 48 h the receptor levels were below basal. Stimulation of NP receptors by eCG was paralleled by activation of both ovarian ANP and C-type natriuretic peptide (CNP) gene expression. ANP mRNA increased as early as 1 h after eCG injection and remained elevated up to 6 h. CNP mRNA increased at 2 h after eCG injection, peaked (5-fold) at 6 h, and remained elevated 48 h later, a stage at which follicular maturation continues. Incubation of ovaries with ANP significantly decreased eCG-induced estradiol level, indicating the functionality of the ovarian NP system. These results implicate the NP system in the induction and maintenance of fluid balance in the rapidly developing ovarian follicle.

Animals↗

PubMed1998

Impairing follicle-stimulating hormone (FSH) signaling in vivo: targeted disruption of the FSH receptor leads to aberrant gametogenesis and hormonal imbalance.

Pituitary gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone stimulate the gonads by regulating germ cell proliferation and differentiation. FSH receptors (FSH-Rs) are localized to testicular Sertoli cells and ovarian granulosa cells and are coupled to activation of the adenylyl cyclase and other signaling pathways. Activation of FSH-Rs is considered essential for folliculogenesis in the female and spermatogenesis in the male. We have generated mice lacking FSH-R by homologous recombination. FSH-R-deficient males are fertile but display small testes and partial spermatogenic failure. Thus, although FSH signaling is not essential for initiating spermatogenesis, it appears to be required for adequate viability and motility of the sperms. FSH-R-deficient females display thin uteri and small ovaries and are sterile because of a block in folliculogenesis before antral follicle formation. Although the expression of marker genes is only moderately altered in FSH-R -/- mice, drastic sex-specific changes are observed in the levels of various hormones. The anterior lobe of the pituitary gland in females is enlarged and reveals a larger number of FSH- and thyroid-stimulating hormone (TSH)-positive cells. The phenotype of FSH-R -/- mice is reminiscent of human hypergonadotropic ovarian dysgenesis and infertility.

Animals↗

PubMed1998

Is there a true requirement for follicle stimulating hormone in promoting spermatogenesis and fertility in primates?

Although the role of follicle stimulating hormone (FSH) in regulating ovarian function is well accepted, its need in regulating testicular function of the adult continues to be debated. Sertoli cells of all mammals have FSH receptors and are known to regulate differentiation and transformation of germ cells to spermatozoa. However, there appear to be species and age differences in the way in which FSH regulates spermatogenesis. An attempt has been made in the current paper to examine critically the newer data available in support of and against the concept that FSH is required to regulate spermatogenesis and fertility of the primate. As there is no evidence to indicate that testicular function in monkeys and humans is regulated differently, the information available using the monkey as the experimental surrogate model is discussed in some depth. These are correlated, wherever feasible, to the newer information emerging from clinical studies. It appears from these studies that in the primate (including humans) FSH, besides promoting quantitative spermatogenesis leading to production of a normal number of spermatozoa, has a critical role in regulating spermiogenesis, the process that controls the formation of normal fertilizable mature spermatozoa. While the requirement for FSH in promoting fertility in the male monkey is reasonably well established, in humans the evidence currently available in favour of the concept is still circumstantial and more work needs to be done to establish the hypothesis beyond any doubt.

Animals↗

PubMed1997

Molecular cloning, structure, and expression of a testicular follitropin receptor with selective alteration in the carboxy terminus that affects signaling function.

During the molecular cloning of the ovine testicular follicle-stimulating (FSH) receptor that couples to the Gs-type effector systems, we discovered novel cDNA clones that were highly homologous. Some of these clones contained an insert of 1,584 bp, which consisted of a divergent 3' region spliced with a 5' region that was identical to nucleotides 724-1,924, forming part of the 9th and 10th exons, of the coding region of the ovine FSH receptor gene. The prominence of alternately spliced clone, which suggested important functional implications, prompted this detailed investigation. Screening of the library by polymerase chain reaction and Northern analysis of testicular messenger RNA with a specific ribo-probe directed to the divergent 3' region of this transcript suggested existence of a full-length transcript of roughly 2.4 kb size. The cDNA was assembled and characterized for its structure. The predicted full-length sequence consisting of nucleotides -121-1,924 of the ovine FSH receptor and the novel 3' region (nucleotides 1,925-2,307) encoded a protein of 670 amino acids containing the entire extracellular and transmembrane domains of the ovine FSH receptor. However, a frame-shift in the coding sequence at the point of divergence resulted in a shorter (40 residues vs. 65 for ovine FSH receptor) C-terminus with three cysteine residues and a reduced number of potential phosphorylation sites. Two of the cysteine residues were adjacent and are apparently potential double palmitoylation sites compared to the single site present in the Gs coupled ovine FSH receptor. Stable expression of this novel transcript in human embryonic kidney (HEK 293) cells revealed the complete absence of cyclic AMP inducible functions, but presence of specific hormone binding activity on plasma membranes and prominent cell surface immunostaining by antireceptor antiserum. There was no alteration in hormone binding specificity because the structurally analogous luteinizing hormone (LH) did not bind to the receptor. The loss of cyclic AMP stimulation in the transfected cells was completely opposite to the properties of the cells expressing the active wild-type receptor. When cells expressing active receptors were cotransfected with the altered FSH receptor cDNA, hormone action was inhibited, suggesting that it could be functioning as a dominant negative receptor. The existence of this ovine FSH receptor with an altered carboxyl terminus predicts the utilization of an alternative splicing mechanism for regulation of receptor expression, signalling and gonadal function. Our study reveals that the modular structure of the FSH receptor gene generates motifs that allows coupling to different effectors. This could become a common feature for all glycoprotein hormone receptors.

Adenylyl Cyclases↗

PubMed1997

Alternative splicing converts the G-protein coupled follitropin receptor gene into a growth factor type I receptor: implications for pleiotropic actions of the hormone.

Pituitary follitropin (FSH) has pleiotropic actions on gonads, but it is not certain if all these events are mediated by a single receptor. A single gene for the FSH receptor undergoes extensive alternate splicing generating multiple transcripts, and several of these have been cloned and characterized from the sheep testis. In this study we have investigated the expression in HEK (human embryonic kidney) 293 cells of a cloned cDNA encoding the first eight exons of the FSH receptor along with a carboxyterminal extension that contributed a hypothetical single transmembrane domain. This cDNA, which lacked the conventional seven transmembrane motif of the full-length 695 residue wild-type receptor protein, was also efficiently expressed on the cell surface and exhibited high affinity and specificity for FSH binding. LH did not compete for FSH binding indicating that these structures contained all the motifs necessary for specific hormone recognition. Following hormone binding and affinity crosslinking the deduced M(r) of the expressed receptor was compatible with dimer formation. The expression of these altered FSH receptors on the cell surface was confirmed by immunohistochemistry, which revealed punctate labeling in a pattern comparable to that shown by cells transfected by wild-type receptor cDNA. Addition of FSH stimulated 3H-thymidine incorporation in transfected cells in a biphasic manner. By performing RT-PCR we could show that similar altered receptor transcripts were present in both the ovary and testis. Our results reveal for the first time that the seven transmembrane structure of FSH-receptor is not absolutely necessary for cell surface expression and hormone binding provided other compensating motifs are present in the protein structure for membrane insertion. Some of these features are typical of growth factor receptors. Our investigations also demonstrate that alternate splicing of the FSH receptor gene provides a mechanism for creating receptor diversity and suggest that multiple receptors could be involved in regulation of hormone action.

Alternative Splicing↗

PubMed1997

Characterization of the 5' flanking region and potential control elements of the ovine follitropin receptor gene.

In the preceding two reports, we presented evidence for the structure and functional characteristics of two different, yet related variants of the sheep testicular follicle-stimulating hormone receptor (oFSH receptor) cDNA. To shed further light on the structural basis of the formation of such receptor forms with different motifs and the eventual understanding of gene regulation, we initiated studies to clone the gene. An 8 kb EcoR I fragment containing the exon-1 and 5' flanking sequence was cloned and characterized from among the 14 clones that were isolated from the genomic library. Although not all other clones were fully characterized, we believe that the entire gene of 85-100 kb has been secured as we adopted a successive screening strategy to accommodate currently known alternatively spliced variants of the receptor in this species. This has led us to propose a revised model that includes an 11th exon for the oFSH receptor gene. The 11th exon that lies beyond the currently postulated 10th exon contributes important DNA sequence that results in two different structural/functional motifs. One creates a dominant negative receptor and the other leads to the formation of a growth factor type I receptor for the hormone. In the 2.1 kb 5'-upstream region, there are a number of potentially interesting regulatory elements that resemble sites for estrogen response element (ERE-like), CRE, and orphan receptor (SF-1/ NGF I-A) transcription factors among others. Other interesting features include the presence of potential germ cell specific and methylation sites. By performing primer extension with testicular RNA, we could identify a single major transcription start site at -163 relative to +1ATG. The availability of the structure of FSH-receptor gene in this domestically important seasonal breeder could spur investigations into the control of receptor gene expression.

Alternative Splicing↗

PubMed1997

Debunking hCG.

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AIDS-Related Opportunistic Infections↗

PubMed1997

Immunization of male bonnet monkeys (M. radiata) with a recombinant FSH receptor preparation affects testicular function and fertility.

Immunization of proven fertile adult male monkeys (n=3) with a recombinant FSH receptor protein preparation (oFSHR-P) (representing amino acids 1-134 of the extracellular domain of the receptor Mr approximately 15KDa) resulted in production of receptor blocking antibodies. The ability of the antibody to bind a particulate FSH receptor preparation and receptors in intact granulosa cells was markedly (by 30-80%) inhibited by FSH. Serum T levels and LH receptor function following immunization remained unchanged. The immunized monkeys showed a 50% reduction (p<0.001) in transformation of spermatogonia(2C) to primary spermatocytes (4C) as determined by flow cytometry and the 4C:2C ratio showed a correlative change (R 0.81, p<0.0007) with reduction in fertility index (sperm counts X motility score). Breeding studies indicated that monkeys became infertile between 242-368 days of immunization when the fertility index was in the range of 123+/-76 to 354+/-42 (compared to a value of 1602+/-384 on day 0). As the effects observed are near identical to that seen following immunization with FSH it is suggestive that oFSHR-P can substitute for FSH in the development of a contraceptive vaccine.

Animals↗

PubMed1997

Low molecular weight components but not dimeric HCG inhibit growth and down-regulate AP-1 transcription factor in Kaposi's sarcoma cells.

Kaposi's sarcoma, a sexually dimorphic disease inflicting high mortality in AIDS, remains at present without effective treatment. A recent report (Nature 375:64, 1995) showed that the placental glycoprotein hormone, human chorionic gonadotropin (HCG), and surprisingly its beta subunit, inhibit tumorigenicity and metastasis of Kaposi's sarcoma cells in mice xenografts. The anti-KS efficacy of a commercial HCG was subsequently demonstrated in clinical trials. Experimental data presented herein confirm that commercial HCG preparations (known to be about 25% pure) display significant inhibitory action in a dose-dependent manner. However, pure and biologically active HCG has no effect on Kaposi's sarcoma growth in culture. In fact, incubation of Kaposi's sarcoma cells with either one of four different well characterized preparations of pure HCG dimer or any of its two subunits did not alter cellular proliferation suggesting that a contaminant (or degradation product) may be the active agent. Commercial HCG preparations were subfractionated based on molecular size and each fraction was tested with respect to inhibition of KS cell growth, HCG radioreceptor binding and steroidogenic bioactivity. Results demonstrate that the anti-KS activity resides among low molecular weight components, and not in bona fide (macromolecular) HCG. Our study indicates that HCG activity and anti-KS action are separable. Interestingly, the active components in the crude HCG markedly down-regulate AP-1, a complex of transcription factors of the immediate-early response genes associated with cell growth. We conclude that, as yet unidentified molecules, present in the commercial HCG preparations, are responsible for the growth inhibitory effects presumably via the AP-1 signalling pathway.

Acquired Immunodeficiency Syndrome↗

PubMed1997

Ontogeny of FSH receptor messenger ribonucleic acid transcripts in relation to FSH secretion and testicular function in sheep.

The role of alternative splicing of the FSH receptor gene in the generation of FSH receptor proteins and testicular function remains an enigma. To address this issue, this investigation was conducted to determine variations in the expression of alternate FSH receptor mRNA transcripts in relation to changes in FSH release, hormone binding activity and testicular function during pubertal development of ram lambs from two genotypes of sheep (Romanov and a cross between Booroola x DLS) with different sexual precocity. Serum 17 beta-estradiol and testosterone concentrations were used as indices of Sertoli cell and testicular function. The results indicated that increases in Sertoli cell and testicular function normally seen during pubertal development are accompanied by age-dependent reductions in concentration of functional FSH receptors, as determined by binding of iodinated FSH to testicular membrane preparations. During the course of these changes, FSH release was either maintained at a steady level in Romanov lambs or it was gradually reduced in the Booroola x DLS cross, thus indicating that the testis had become more responsive to hormonal signal. This acquisition of heightened sensitivity was also associated with contrasting changes in the level of expression of FSH receptor mRNA transcripts. For both geno-types of sheep, 5 distinct species of mRNA transcripts of approximately 1.1, 1.5, 2.0, 2.5 and 6.5 kb were highly expressed from 11 to 22 weeks of age. Amongst these transcripts, the 1.1 kb molecular species was the most abundant. Specific probing for a previously cloned transcript called 151A1 representing the first 4 exons of the FSH receptor gene revealed a paradoxical increase in the level of expression from 11 weeks up to a maximum at 18-22 weeks of age for both genotypes. Collectively, the results indicated that contrasting changes in the production of specific alternatively spliced mRNA transcripts may mediate changes in FSH receptor expression which apparently accounts for the augmentation in sensitivity and function of the testis during pubertal development. Furthermore, the data provide the first important indication that the novel truncated transcript (151A1), which predictably encodes a soluble protein of either intra- or extracellular fate, could be physiologically relevant.

Animals↗

PubMed1997

Recognition of follicle stimulating hormone (alpha-subunit) by a recombinant receptor protein domain coded by an alternately spliced mRNA and expressed in Escherichia coli.

To assess the functional significance of putative proteins encoded by alternately spliced mRNA of the sheep testicular FSH receptor, a short form cDNA comprising of the first four exons (117 residues mature protein) was engineered for expression in Escherichia coli. The expressed protein of molecular mass 15 kDa was purified to homogeneity and verified by reaction with an antibody against a synthetic peptide sequence unique to the amino (N)-terminal region FSH receptor. The purified FSH receptor domain protein bound 125I-labeled hFSH in a ligand blot on polyvinylidine difluoride membranes. Further analyses by slot blot revealed high affinity of the immobilized protein with significant reaction at 10 pmol. As the immobilized receptor protein also reacted with structurally related hormones (125I-labeled LH/125I-labeled human chorionic gonadotropin), we confirmed that interaction most probably occurred via the common alpha-subunit of these glycoprotein hormones. Our results reveal that this N-terminal portion of the FSH receptor contain(s) major site(s) for hormone recognition that could be mediated via the alpha-subunit. A rabbit antibody to the receptor inhibited FSH action in receptor bearing cells, revealing the utility of such recombinant FSH receptor protein(s) for modulation of hormone action.

Alternative Splicing↗

PubMed1996

Follitropin signal transduction: alternative splicing of the FSH receptor gene produces a dominant negative form of receptor which inhibits hormone action.

We have studied the functional properties of an alternately spliced form of sheep testicular FSH receptor cDNA that codes for a protein similar to a previously described active receptor but differs in the carboxy terminus in sequence and is also shorter by 25 residues. The receptor expressed in HEK 293 cells fails to activate adenylate cyclase. Cotransfection of stably expressing cells bearing FSH receptor that activates (Gs) adenylate cyclase with the altered receptor cDNA abrogates hormone response. In cells expressing this cDNA. FSH also inhibited cyclic AMP accumulation induced by non hormonal agents such as forskolin and cholera toxin which bypass the receptor. We propose that this altered receptor is a dominant negative receptor which may be coupled to G1 protein(s) or other inhibitory mechanisms.

Adenylyl Cyclases↗

PubMed1995

Bacterial expression of human chorionic gonadotropin alpha subunit: studies on refolding, dimer assembly and interaction with two different beta subunits.

Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that have a common alpha subunit but differ in their hormone-specific beta subunit. The common alpha subunit contains two asparagine (N)-linked oligosaccharides. To study the function of carbohydrates on in vitro refolding of alpha subunit and dimer assembly, we generated recombinant non-glycosylated hCG alpha subunit (rNGl-hCGalpha) from E. coli. The expression vector was constructed by inserting hCGalpha cDNA coding for the mature form in-frame into a pQE-30 vector, which contains a 6 x His sequence immediately before the 5'-end of hCGalpha cDNA for subsequent purification of rNG-hCGalpha. The rNG-hCGalpha expressed in inclusion bodies was efficiently purified by immobilized metal chelate affinity chromatography on Ni-NTA resin. SDS-PAGE, solid-phase binding assay and immunoblotting demonstrated the expression of rNG-hCG. Its alpha molecular weight on SDS-PAGE was 14.7 kDa under reducing conditions and 15 kDa for a monomer accompanied with some higher molecular weight oligomer under non-reducing conditions. Reconstitution of rNG-hCGalpha with native hCGbeta and oFSHbeta occurred in very low yield under standard conditions. However, the oxidation-reduction system cystamine (1.34 mM) and cysteamine (7.3 mM) facilitated both the refolding of rNG-hCGalpha and reconstitution of rNG-hCGalpha with native hCGbeta to regain partially correct conformation. These were revealed by conformationally sensitive antibody and receptor binding assays. Cystamine and cysteamine were more effective in the recombination of rNG-hCGalpha with oFSHbeta as indicated by a 22-36-fold decrease in the amount required to cause a 50% competitive inhibition in radioreceptor assay. They have no effect on assembly of rNG-hCGalpha with oLHbeta. Our results suggest the carbohydrate moieties confer greater conformational flexibility to the backbone of the beta subunit and the relative rigidity of the beta subunit may serve as a conformational template of the alpha subunit. The present approach has made it possible to prepare the non-glycosylated gonadotropin alpha subunit in adequate amounts for further study on their biological and topographical features in complete absence of carbohydrate.

Amino Acid Sequence↗

PubMed1994

Topographical differences in human choriogonadotropin antagonist as revealed by monoclonal antibodies.

Human choriogonadotropin (hCG), a highly glycosylated hormone loses its biological activity following deglycosylation. The deglycosylated hormone (DG-hCG) binds to gonadal cell receptors without causing complete signal transduction. In order to analyze the molecular features of DG-hCG, we have prepared and studied five monoclonal antibodies (Mab). Two DG-hCG Mabs were highly specific while three others showed some but low cross-reactivity with hCG. Only those that recognized hCG albeit weakly, could inhibit binding of 125I-hCG to receptors or hCG action in MA-10 (mouse Leydig tumor cells). All five Mabs were highly reactive with the free alpha subunit. As these alpha subunit favoring antibodies easily recognized DG-hCG but not the intact hCG configuration, we suggest that loss of sugars in alpha and beta subunits of DG-hCG was responsible for these alterations. Thus, in the molecular topography of DG-hCG, the orientation of the alpha subunit would be different from that in the native hormone and this may be further perturbed following binding to the specific receptor, leading to inefficient coupling to the signal transducing machinery.

Animals↗

PubMed1994

Isolation and characterization of distinct bioactive forms of LH from male buffalo pituitaries: differences localized to their alpha subunits.

The isolation of highly purified forms of pituitary LH from Egyptian male (Nile) buffaloes is described. The total LH content (receptor binding activity) which was approximately 30 to 50 fold higher than FSH in the pituitary could be divided into three pools based upon fractionation patterns on a cation exchanger. The acidic fraction which also contained FSH was not purified to homogeneity. A basic fraction (bu-LH-2; 300 mg/kg anterior pituitary) and a very basic fraction (bu-LH-3; 80 mg/kg) were both highly purified and free of FSH activity as tested by specific FSH receptor and immunoassays. The basic buffalo LH fraction, bu-LH-2, was as active as highly purified ovine LH (oLH). The most basic form of buffalo LH, bu-LH-3, was, however, about twice as active as highly purified oLH in the in vitro bioassay using mouse Leydig tumour (MA-10) cells. In a receptor binding assay employing 125I-labelled buffalo LH (bu-LH-3) and porcine testicular membranes, the affinity of bu-LH-3 was about five times higher than purified oLH. The M(r) of both forms of purified buffalo LH and subunits was similar to that of oLH. Amino acid composition of buffalo LH was also very similar to oLH except for small differences. Fractionation by fast protein liquid chromatography on Mono-Q columns revealed further evidence of microheterogeneity in each of the pools of buffalo LH with bu-LH-3 exhibiting a predominant single component. By reverse-phase high-pressure liquid chromatography analysis we have localized differences in the two purified isoforms of male buffalo LH to the alpha subunit. It is suggested that differences in biological potencies could be due to variations in terminal glycosylation and/or differences in branching of this subunit which is known to be important for signal transduction.

Amino Acids↗

PubMed1993

Cloning of alternately spliced mRNA transcripts coding for variants of ovine testicular follitropin receptor lacking the G protein coupling domains.

We report the cloning and characterization of two alternately spliced forms of ovine testicular follitropin receptor mRNA. A smaller receptor cDNA (151 A1) of 727 bp codes for a possible soluble receptor protein of 134 amino acids arising from exons 1-4 of the full length receptor. The 1.1 Kb cDNA clone (HK 18) extending up to the 8th exon codes for a mature protein of 259 amino acids with a single membrane spanning domain predicted by hydropathy analysis. As these structures account for 34% and 61% respectively of the extracellular domain of the full length receptor, we suggest that their putative protein products are likely to possess moderate or high affinity binding sites of physiological significance.

Alternative Splicing↗

PubMed1993

Cytotoxic activity of lutropin-gelonin conjugate in mouse Leydig tumor cells: potentiation of the hormonotoxin activity by different drugs.

A hormonotoxin preparation composed of gelonin, a basic protein of 30,000 Da isolated from the plant Gelonium multiflorum and the luteinizing hormone (LH, lutropin) isolated from the sheep pituitary has been studied for its cytotoxic action on mouse testicular Leydig tumor cells (MA-10 cells). Gelonin modified with 2-iminothiolane and conjugated with hormone modified by N-succinimidyl-3-2-pyridyl dithiopropionate was able to inhibit protein synthesis in Leydig tumor cells. An enhancement of the cytotoxicity of the hormonotoxin was obtained in the presence of drugs like quinacrine, chloroquine, verapamil and monensin. We report that the cytotoxicity of hormonotoxin was enhanced 10-15 times with quinacrine (7.6 microM), chloroquine (29 microM), verapamil (40 microM) and monensin (0.29 microM). While quinacrine, chloroquine and verapamil were not cytotoxic to MA-10 cells for up to 48 h, monensin alone reduced protein synthesis significantly in 48 h. All the drugs studied here inhibited steroidogenic action of the native hormone even at concentrations which were not detrimental to protein synthesis. On the basis of the above studies, we suggest that it may be feasible to develop combination strategies to destroy gonadal cells bearing gonadotropin (LH) receptors. In cells not bearing LH receptors (COS-7 cell line) there was no cytotoxicity either with hormonotoxin alone or in combination with the drugs, suggesting specificity of action.

Animals↗