Clinically nonfunctioning pituitary adenomas are benign neoplasms comprising approximately 25-30% of pituitary tumors. Little is known about the pathogenesis of pituitary neoplasia. Clonal analysis allows one to make the important distinction between a polyclonal proliferation in response to a stimulatory factor versus a monoclonal expansion of a genetically aberrant cell. We investigated the clonal origin of pituitary tumors using X-linked restriction fragment length polymorphisms at the phosphoglycerate kinase and hypoxanthine phosphoribosyl-transferase genes. Restriction enzymes were used to distinguish maternal and paternal X-chromosomes, and combined with a methylation-sensitive restriction enzyme to analyze allelic X-inactivation patterns in six pituitary adenomas. All six tumors showed a monoclonal pattern of X-inactivation. These data indicate that nonfunctioning pituitary adenomas are unicellular in origin, a result consistent with the hypothesis that this tumor type is due to somatic mutation.
J M Alexander, B M Biller, H Bikkal, N T Zervas, A Arnold, A Klibanski
In the course of examining the structure and function of Fc receptors on peripheral blood cells of patients with systemic lupus erythematosus, we identified a patient whose neutrophils did not react with either monoclonal or polyclonal antibodies to Fc receptor III. However, neutrophils from the patient were comparable to neutrophils from healthy controls with respect to their expression of Fc receptor II, complement receptor 1, complement receptor 3, and the phosphatidylinositol-linked, complement regulatory protein, decay-accelerating factor. The abnormality of expression of Fc receptor III was limited to the patient's neutrophils (her natural killer cells reacted normally with anti-Fc receptor III antibodies), and was associated with abnormal recognition and binding of IgG-coated erythrocytes. Analysis of genomic DNA revealed evidence that failure of the patient's neutrophils to express Fc receptor III was most likely due to an abnormality of the gene that encodes this receptor.
M R Clark, L Liu, S B Clarkson, P A Ory, I M Goldstein
Argininemia results from a deficiency of arginase (EC 3.5.3.1), the last enzyme of the urea cycle in the liver. We examined the molecular basis for argininemia by constructing a genomic library followed by cloning and DNA sequencing. Discrete mutations were found on two alleles from the patient, a product of a nonconsanguineous marriage. There was a four-base deletion at protein-coding region 262-265 or 263-266 in exon 3 that would lead to a reading-frame shift after amino acid residue 87 and make a new stop codon at residue 132. The other was a one-base deletion at 77 or 78 in exon 2 that would lead to a reading-frame shift after residue 26 and make a stop codon at residue 31. For confirmation, genomic DNAs from the patient and from her parents were amplified by the polymerase chain reaction method. The patient was shown to be a compound heterozygote, inheriting an allele with the four-base deletion from the father and the other allele with the one-base deletion from the mother. These data seem to be the first evidence of a case of argininemia caused by two different deletion mutations.
Y Haraguchi, J M Aparicio, M Takiguchi, I Akaboshi, M Yoshino, M Mori, I Matsuda
Prolidase deficiency is an autosomal recessive disorder characterized by mental retardation and various skin lesions. Cultured skin fibroblasts were obtained from two independent patients with abnormal prolidase. Using the polymerase chain reaction, we amplified the entire coding region of human prolidase mRNA derived from patients' fibroblasts. Nucleotide sequence analysis of amplified cDNA products revealed a G to A substitution at position 826 in exon 12, where aspartic acid was replaced by asparagine at the amino acid residue 276, in cells from both patients. An analysis of the DNA showed that the substitution was homozygous. An expression plasmid clone containing a normal human prolidase cDNA (pEPD-W) or mutant prolidase cDNA (pEPD-M) was prepared, transfected, and tested for expression in NIH 3T3 cells. Incorporation of pEPD-W and pEPD-M resulted in the synthesis of an immunological polypeptide that corresponded to human prolidase. Active human enzyme was detected in cells transfected with pEPD-W, but not in those transfected with pEPD-M. These results were compatible with our observation of fibroblasts and confirmed that the substitution was responsible for the enzyme deficiency. As active prolidase was recovered in prolidase-deficient fibroblasts transfected with pEPD-W, this restoration of prolidase activity after transfection means that gene replacement therapy for individuals with this human disorder can be given due consideration.
A Tanoue, F Endo, A Kitano, I Matsuda
Autocrine stimulation of the epidermal growth factor receptor (EGF-R), by coexpression of transforming growth factor-alpha (TGF-alpha), causes malignant transformation of some fibroblast cell lines. TGF-alpha and EGF-R are both known to be expressed in colon carcinoma tissue and have been shown coexpressed in colon carcinoma cell lines. TGF-alpha autocrine activation of EGF-R has been suggested as a potential mechanism contributing to abnormal growth control in colon cancer. We now report coexpression of TGF-alpha and EGF-R transcripts in morphologically normal colonic epithelium from five individuals, in colonic adenomas from three individuals, and in a nontumorigenic colon adenoma cell line, VACO-330. Functional studies demonstrate VACO-330 growth is stimulated by exogenous TGF-alpha and is completely abolished by a blocking anti-EGF-R antibody. Autocrine stimulation of EGF-R by TGF-alpha is therefore required for growth of the adenoma cell line. Autocrine stimulation of EGF-R by TGF-alpha does not cause malignant transformation of the colonic epithelial cell. In normal and adenomatous human colon TGF-alpha, via either an autocrine or paracrine mechanism, is likely an important physiologic stimulant of epithelial proliferation.
S D Markowitz, K Molkentin, C Gerbic, J Jackson, T Stellato, J K Willson
The p53 gene initially was thought to be an oncogene, but recent evidence suggests that wild-type p53 can function as a tumor suppressor gene in lung, colon, and breast cancer as well as less common malignancies. This study reports the first identification of intronic point mutations as a mechanism for inactivation of the p53 tumor suppressor gene. Abnormally sized p53 mRNAs found in a small cell and a non-small cell lung cancer cell line were characterized by sequence analysis of cDNA/PCR products, the RNase protection assay and immunoprecipitation. These mRNAs were found to represent aberrant splicing leading to the production of abnormal or no p53 protein. Sequence analysis of genomic DNA revealed that a point mutation at the splice acceptor site in the third intron or the splice donor site in the seventh intron accounts for the abnormal mRNA splicing. In one patient the same intronic point mutation was found in the tumor cell line derived from a bone marrow metastasis and in multiple liver metastases but not in normal DNA, indicating that it occurred as a somatic event before the development of these metastases. These findings further support the role of inactivation of the p53 gene in the pathogenesis of lung cancer and indicate the role of intronic point mutation in this process.
T Takahashi, D D'Amico, I Chiba, D L Buchhagen, J D Minna
The aim of this study was to investigate the susceptibility of human myotubes to lysis by the two major types of cytotoxic effector cells, CD3+CD8+ cytotoxic T cells (CTL) and CD16+CD56+ natural killer (NK) cells. The myoblasts preparations used as target cells were greater than 90% pure as assessed by immunostaining with the Leu19 monoclonal antibody (MAb) that cross-reacts with the neural cell adhesion molecule N-CAM. Allospecific CTL lines were generated from mixed lymphocyte cultures, and freshly isolated allogeneic and autologous peripheral blood cells were used as a source of NK cells. The cytotoxicity was observed under phase optics and by immunoelectron microscopy, and was quantitated with a chromium release assay. Myotubes were efficiently killed by allospecific CTL and by autologous and allogeneic NK cells. The killing by CTL was inhibited with an anti-class I HLA MAb, and the killing by NK cells was inhibited by depleting peripheral blood cells of CD16+ cells with anti-CD16 MAb and complement. The results have important implications for myoblast transplantation, an experimental therapy of muscular dystrophy.
R Hohlfeld, A G Engel
Idiopathic hypoparathyroidism has been reported to occur as an X-linked recessive disorder in two multigeneration kindreds. Affected individuals, who are males, suffer from infantile onset of epilepsy and hypocalcemia, which appears to be due to an isolated congenital defect of parathyroid gland development; females are not affected and are normocalcemic. We have performed linkage studies in these two kindreds (5 affected males, 11 obligate carrier females, and 44 unaffected members) and have used cloned human X chromosome sequences identifying restriction fragment length polymorphisms to localize the mutant gene causing this disorder. Our studies established linkage between the X-linked recessive idiopathic hypoparathyroid gene (HPT) and the DXS98 (4D.8) locus, peak LOD score = 3.82 (theta = 0.05), thereby mapping HPT to the distal long arm of the X chromosome (Xq26-Xq27). Multilocus analysis indicated that HPT is proximal to the DXS98 (4D.8) locus but distal to the F9 (Factor IX) locus, thereby revealing bridging markers for the disease. The results of this study will improve genetic counseling of affected families, and further characterization of this gene locus will open the way for elucidating the factors controlling the development and activity of the parathyroid glands.
R V Thakker, K E Davies, M P Whyte, C Wooding, J L O'Riordan
Studies were performed to examine interactions between the adenylyl cyclase (AC) and phospholipase C (PLC) signaling systems in cultured rat inner medullary collecting duct cells. Stimulation of AC by either arginine vasopressin (AVP) or forskolin or addition of exogenous cAMP inhibits epidermal growth factor (EGF)-stimulated PLC. This inhibition is mediated by activation of cAMP-dependent kinase as it is prevented by pretreatment with the A-kinase inhibitor, N-[2-(methylamino)ethyl]-5-isoquinoline-sulfonamide (H8) but not by the C-kinase inhibitor, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7). Exposure to EGF eliminates AVP-stimulated cAMP generation. This is not mediated by a cyclooxygenase product as inhibition by EGF is observed even in the presence of the cyclooxygenase inhibitor, flurbiprofen. Inhibition by EGF is not due to an increase in inositol trisphosphate (IP3) as exposure of saponin-permeabilized cells to exogenous IP3 is without effect. Inhibition by EGF is prevented by pretreatment with the C-kinase inhibitor, H7, but not by the A-kinase inhibitor, H8. Exposure to the synthetic diacylglycerol (DAG), dioctanoylglycerol, also inhibits AVP-stimulated AC activity; therefore, inhibition by EGF is due to activation of protein kinase C. Thus, in cultured rat inner medullary collecting duct cells, cAMP and DAG function as mutually inhibitory second messengers with each impairing formation of the other.
I Teitelbaum
We have examined the effects of several PGs on the synthesis and release of the atrial natriuretic peptide (ANP) in vivo and in vitro. PGF2 alpha infusion in anesthetized rats resulted in a significant increase in plasma immunoreactive (ir) ANP levels in vivo despite effecting only modest changes in hemodynamics. The PGs were also effective at promoting irANP secretion in primary cultures of neonatal rat atrial and ventricular cardiocytes. PGF2 alpha increased irANP release with half-maximal induction seen at approximately 10(-8) M; PGE2 was somewhat less effective and prostacyclin (PGI2) was without effect. The PGs also increased ANP mRNA levels in these cells, suggesting that these agents exert a major effect on the synthesis as well as the secretion of the prohormone. Transient expression analysis of atrial cells transfected with 2,500 bp of human (h) ANP 5' flanking sequence linked to a chloramphenicol acetyltransferase (CAT) reporter demonstrated that PGF2 alpha (10(-5) M) increased hANP promoter activity approximately twofold relative to the control. PGF2 alpha had no effect on the promoterless control (pSV0-lamin CAT). Treatment of cultured atriocytes with high concentrations of a cyclooxygenase inhibitor resulted in a significant suppression of ANP secretion in vitro and a truncation of the plasma ANP response to volume infusion in vivo. Taken together these studies support a role for PGs as regulators of cardiac ANP synthesis and secretion, and suggest an additional mechanism whereby eicosanoids may act to control cardiovascular and renal homeostasis.
D G Gardner, H D Schultz
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