[Inhibin and ovarian cancer]. / Ingibin i rak iaichnikov.

Previous observations from our laboratory have demonstrated that the levels of immunoreactive inhibin (ir-inh) are elevated in almost all patients with granulosa cell tumors and in the majority of postmenopausal women with mucinous ovarian cancers. The present report confirms these findings in a larger group of post-menopausal women. Immunohistochemistry for the inhibin alpha. beta A and beta B sununits shows predominantly epithelial staining in granulosa cell tumors and in the majority of mucinous cancers. Serous cystadenocarcinomas also frequently show positive staining. Studies seeking to identify G alpha i-2 or FSH receptor mutations have provided negative results in contrast to other reports. Further studies of the roles of the inhibin-related family of peptides in ovarian cancer diagnosis and monitoring are clearly indicated.

Localization of a new prostate-specific antigen-related serine protease gene, KLK4, is evidence for an expanded human kallikrein gene family cluster on chromosome 19q13.3-13.4.

The human tissue kallikrein (KLK) family of serine proteases, which is important in post-translational processing events, currently consists of just three genes-tissue kallikrein (KLK1), KLK2, and prostate-specific antigen (PSA) (KLK3)-clustered at chromosome 19q13. 3-13.4. We identified an expressed sequence tag from an endometrial carcinoma cDNA library with 50% identity to the three known KLK genes. Primers designed to putative exon 2 and exon 3 regions from this novel kallikrein-related sequence were used to polymerase chain reaction-screen five cosmids spanning 130 kb around the KLK locus on chromosome 19. This new gene, which we have named KLK4, is 25 kb downstream of the KLK2 gene and follows a region that includes two other putative KLK-like gene fragments. KLK4 spans 5.2 kb, has an identical genomic structure-five exons and four introns-to the other KLK genes and is transcribed on the reverse strand, in the same direction as KLK1 but opposite to that of KLK2 and KLK3. It encodes a 254-amino acid prepro-serine protease that is most similar (78% identical) to pig enamel matrix serine protease but is also 37% identical to PSA. These data suggest that the human kallikrein gene family locus on chromosome 19 is larger than previously thought and also indicate a greater sequence divergence within this family compared with the highly conserved rodent kallikrein genes.

Inhibin and ovarian cancer.

Previous observations from our laboratory have demonstrated that the levels of immunoreactive inhibin (ir-inh) are elevated in almost all patients with granulosa cell tumours and in the majority of postmenopausal women with mucinous ovarian cancers. The present manuscript confirms these findings in a larger group of postmenopausal women. Immunohistochemistry for the inhibin alpha, betaA and betaB subunits shows predominantly epithelial staining in granulosa cell tumours and in the majority of mucinous cancers. Serous cystadenocarcinomas also frequently show positive staining. Studies seeking to identify G alpha(i-2) or FSH receptor mutations have provided negative results in contrast to other reports. Further studies of the roles of the inhibin-related family of peptides in ovarian cancer diagnosis and monitoring are clearly indicated.

No evidence of a role for mutations or polymorphisms of the follicle-stimulating hormone receptor in ovarian granulosa cell tumors.

The molecular pathogenesis of granulosa cell tumors of the ovary is not understood, although recent studies have shown that immunoreactive inhibin secretion by these tumors may be used as a tumor marker. Granulosa cell tumors exhibit many features of normal granulosa cells, including a response to FSH and inhibin secretion. FSH levels are suppressed in patients with inhibin-secreting granulosa cell tumors, suggesting FSH-independent growth of these tumors. Activating mutations of the FSH receptor might, therefore, be involved in tumorigenesis. We sought to identify mutations in the FSH receptor genes of these tumors using PCR to amplify the exon encoding the transmembrane and cytoplasmic domains from the tumor DNA. Analysis of the amplicons for single strand conformational polymorphisms and direct sequencing confirmed a previously reported polymorphism in the C-terminal region of the receptor, but did not identify tumor-specific missense mutations and/or polymorphisms. In addition, ribonucleic acid from 3 granulosa cell tumors was used to confirm expression of the FSH receptor; expression was unexpectedly also observed in several ovarian mucinous cystadenocarcinomas used as controls. In conclusion, our failure to identify activating mutations of the FSH receptor in 15 granulosa cell tumors argues against a role for the FSH receptor in tumorigenesis and suggests that some subsequent component of this signal transduction pathway may be activated.

Kallikrein gene expression in human pituitary tissues.

OBJECTIVE: The glandular kallikreins are a family of enzymes involved in the post-translational processing of polypeptides. Three family members have been characterized in the human: glandular kallikrein (KLK1) and two genes expressed in the prostate, KLK2 or hGK1 and KLK3 or prostate specific antigen (PSA). Both kallikrein immunoreactivity and KLK1 mRNA have been detected in the rat anterior pituitary and oestrogen induced prolactinomas. Immunoreactive kallikrein has also been reported in prolactin secreting pituitary tumours in humans. In this study we wished to determine whether KLK1 and/or other KLK genes were expressed in human pituitary tumours. DESIGN AND PATIENTS: Retrospective analysis of KLK gene expression in pituitary tissue obtained at surgery from 11 patients with a range of pituitary syndromes, 10 of which were tumour induced and included four prolactinomas. Three normal pituitaries, obtained at necropsy, were also analysed. MEASUREMENTS: Pituitary total RNA was subjected to both Northern blot analysis, with KLK1 and KLK2 cDNA probes, and KLK-specific reverse transcriptase -- polymerase chain reaction (RT-PCR). RESULTS: No KLK1 gene expression was detected on Northern blot analysis although expression of PRL, GH and pro-opiomelanocortin (POMC) was variously detected. KLK RT-PCR coupled with Southern blot analysis using oligonucleotide probes specific for each of the three KLK genes was positive in many tumours but with varying levels and differential expression. Overall, KLK1 was the most abundant with KLK3 the least. The identity of KLK1 and KLK2 was confirmed by sequencing the PCR products. There was no obvious correlation between KLK1 and hormone gene expression; KLK1 was present in PRL positive and negative tumours and in normal pituitary tissue. CONCLUSIONS: We have demonstrated a low level of expression of KLK1 as well as KLK2 and KLK3 in the human pituitary. The expression of KLK2 and KLK3 in the pituitary is a novel finding. The lack of correlation between PRL and KLK1 gene expression is an unexpected finding suggesting that the interaction between these factors may be more complex in the human pituitary than in the rodent model.

Pseudohypoaldosteronism: molecular characterization of the mineralocorticoid receptor.

Mineralocorticoid resistance (pseudohypoaldosteronism) is a rare condition first described in 1958 and associated with failure to thrive, salt wasting, and dehydration in infancy. In the index case it has previously been shown that binding of aldosterone to mineralocorticoid receptors in peripheral blood lymphocytes is absent; here, we report results of the molecular characterization of the mineralocorticoid receptor in this patient. Genomic DNA extracted from peripheral blood lymphocytes was subjected to Southern blot analysis after digestion with various restriction enzymes. There was no evidence of a major gene rearrangement or deletion. Oligonucleotide primers were designed on the basis of the published human complementary DNA sequence to cover the entire open reading frame of the mineralocorticoid receptor (MR). Total messenger ribonucleic acid (RNA) from lymphocytes was subjected to reverse transcription and amplification using the reverse transcriptase-polymerase chain reaction; the resulting fragments were then purified, subcloned, and sequenced. The patient showed no abnormality in the complementary DNA sequence corresponding to the open reading frame of the MR molecule compared with the published sequence. In addition, semiquantitative assessment of the patient's MR messenger RNA based on the reverse transcriptase-polymerase chain reaction technique suggested that he was producing MR RNA in roughly normal quantities. The mechanism of mineralocorticoid resistance in this case, therefore, remains uncertain, and the possibility must be considered that the underlying abnormality is not in the MR gene, but in an independent gene acting through yet to be characterized processes.

Isolation of a rat amiloride-binding protein cDNA clone: tissue distribution and regulation of expression.

Sodium transport across high-resistance epithelia involves both an apical amiloride-sensitive sodium channel and the basal Na(+)-K(+)-ATPase pump. Aldosterone regulates sodium transport by increasing the sodium permeability of the sodium channels. To study further the regulation of gene expression in sodium-transporting epithelia by corticosteroids, we have cloned an amiloride-binding protein (ABP) cDNA from rat descending colon and kidney. Identical 311 nucleotide cDNAs were amplified from both rat descending colon and kidney, and the predicted amino acid sequence exhibited 83% homology to the equivalent region of the human peptide sequence. Use of this cDNA as a probe resulted in detection of a transcript in both the small and large bowel, thymus, and seminal vesicle. The latter tissue exhibited the highest level of rat ABP expression. Low to undetectable levels of rat ABP were expressed in the descending colon and kidney. No regulation of rat ABP by either class of corticosteroids was observed. Levels of ABP were low at birth and increased gradually to adult levels just before weaning in the bowel. The distribution of rat ABP is not as would be predicted for an aldosterone-induced gene and is thus unlikely to be a component of the amiloride-sensitive electrogenic sodium channel.

Ileal glucagon gene expression: ontogeny and response to massive small bowel resection.

Massive small bowel resection with re-anastomosis of the residual jejunum and terminal ileum results in marked adaptive responses. Various luminal and humoral factors have been implicated in the adaptive response, which may be analogous to the changes occurring in the ileum in the postnatal growth phase. Enteroglucagon, which is synthesized in the L cells of the intestinal mucosa, is thought to be an important humoral factor in this response. In this study, the levels of glucagon gene expression in the rat ileum both after massive small bowel resection and during development are examined. Glucagon messenger RNA levels are increased threefold as part of the adaptive response; the increase is maximal at 2 days and is at least partly dependent on luminal nutrition. Levels of glucagon messenger RNA in the developing ileum increase in the postnatal period until weaning when they decrease somewhat before gradually reaching adult levels.

Mineralocorticoid receptor gene expression in the gastrointestinal tract: distribution and ontogeny.

The gastrointestinal tract is a well characterized target tissue for aldosterone, where it regulates electrolyte transport, particularly in the descending colon. Previous studies have demonstrated the presence of aldosterone receptors in gastrointestinal tissues. We have used specific cRNA probes for the rat mineralocorticoid receptor to explore both the distribution and ontogeny of mineralocorticoid receptor gene expression in the gastrointestinal tract. Mineralocorticoid receptor gene expression is found throughout the small and large intestine, but is absent from the stomach. The highest levels are observed in the distal colon, and significant expression is found in the duodenum; in both tissues levels of expression are higher than those in kidney. In both the developing duodenum and colon, mineralocorticoid receptor gene expression precedes the development of the full physiological response to aldosterone. These findings emphasise the colon as an important target tissue for aldosterone, and raise the question of potential roles for aldosterone in the duodenum.

Colonic sodium-potassium adenosine triphosphate subunit gene expression: ontogeny and regulation by adrenocortical steroids.

Mineralocorticoids and glucocorticoids exhibit overlapping but distinct effects on transepithelial sodium transport in the descending colon. Na,K-ATPase, the major sodium pump, has been variously reported to be regulated by one or both classes of steroids. The present studies explore the ontogeny and steroidal regulation of Na,K-ATPase alpha- and beta-subunit mRNA levels in the descending colon. In descending colon, subunit mRNA levels are low before birth, increasing to reach adult levels at approximately day 25. Dexamethasone treatment caused a rapid dose-dependent increase in colonic Na,K-ATPase subunit mRNA levels. The specific glucocorticoid RU26988 also increased subunit mRNA levels. Aldosterone administration, at doses adequate to yield a profound antinatriuresis, did not alter subunit mRNA levels. Carbenoloxone sodium produced an approximately 3-fold increase in subunit mRNA levels in intact but not adrenalectomized rats. We have demonstrated that Na,K-ATPase subunit gene expression is: 1) low in the fetal colon but achieves plateau levels by day 25; 2) acutely regulated by corticosteroids via type II rather than type I receptors; and 3) increased by carbenoxolone sodium, presumably as a result of increased occupancy of the type II receptor by corticosterone.

Kallikrein-gene expression in the rat gastrointestinal tract.

The serine proteinase glandular kallikrein has been demonstrated in the gastrointestinal tract, although there is some doubt as to whether it is synthesized there or derives from exocrine-gland secretions. Using a rat pancreatic kallikrein cRNA probe we have demonstrated kallikrein-like gene expression in the corpus, duodenum, jejunum, ileum, caecum and colon, and compared the pattern of expression with that of the gastrointestinal peptides somatostatin, gastrin and glucagon. In addition, using a panel of oligonucleotide probes specific for various members of the rat kallikrein-gene family, we have shown that the kallikrein-like gene expressed appears to be expressed as true kallikrein.

Somatostatin gene expression in the thymus gland.

A complex pattern of interactions appears to exist between the immune and neuroendocrine systems. Recently, vasopressin, oxytocin and vasoactive intestinal peptide have been isolated from the thymus. Using a rat somatostatin antisense RNA probe we have demonstrated expression of the somatostatin gene in the rat thymus. Furthermore, we have shown that the levels of thymic somatostatin mRNA exhibit a bell-shaped response to dexamethasone administration. Lipocortin I and II antisense RNA probes have been used as a positive control for the effects of the dexamethasone. We would suggest that somatostatin acts in the thymus in a paracrine mode to modulate T lymphocyte development.

Kallikrein gene expression in estrogen-induced pituitary tumors.

Anterior pituitary kallikrein-like enzyme activity, immunoreactivity and mRNA levels have previously been shown to be regulated by estrogen, in parallel with prolactin. In this study, we have examined the relationship between kallikrein and prolactin mRNA levels in estrogen-induced pituitary tumors. Treatment of Fischer 344 rats with diethylstilbestrol implants for 3, 5 and 7 weeks produced a dramatic increase in kallikrein mRNA levels and a modest increase in prolactin mRNA levels. These changes were partially reversed by bromocriptine treatment, and completely reversed by bromocriptine plus estrogen withdrawal. Using a panel of oligonucleotide probes specific for various members of the rat kallikrein gene family, we have shown that the kallikrein-like gene expressed appears to be true kallikrein.