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Biomedical subjects

P K Donahoe

Publications and source records attributed to P K Donahoe.

At least 19 recordsLinked to original sources
PubMed2006

Findings from aCGH in patients with congenital diaphragmatic hernia (CDH): a possible locus for Fryns syndrome.

Congenital diaphragmatic hernia (CDH) is a common and often devastating birth defect that can occur in isolation or as part of a malformation complex. Considerable progress is being made in the identification of genetic causes of CDH. We applied array-based comparative genomic hybridization (aCGH) of approximately 1Mb resolution to 29 CDH patients with prior normal karyotypes who had been recruited into our multi-site study. One patient, clinically diagnosed with Fryns syndrome, demonstrated a de novo 5Mb deletion at chromosome region 1q41-q42.12 that was confirmed by FISH. Given prior reports of CDH in association with cytogenetic abnormalities in this region, we propose that this represents a locus for Fryns syndrome, a Fryns syndrome phenocopy, or CDH.

Abnormalities, Multiple↗
PubMed2005

Congenital diaphragmatic hernia and chromosome 15q26: determination of a candidate region by use of fluorescent in situ hybridization and array-based comparative genomic hybridization.

Congenital diaphragmatic hernia (CDH) has an incidence of 1 in 3,000 births and a high mortality rate (33%-58%). Multifactorial inheritance, teratogenic agents, and genetic abnormalities have all been suggested as possible etiologic factors. To define candidate regions for CDH, we analyzed cytogenetic data collected on 200 CDH cases, of which 7% and 5% showed numerical and structural abnormalities, respectively. This study focused on the most frequent structural anomaly found: a deletion on chromosome 15q. We analyzed material from three of our patients and from four previously published patients with CDH and a 15q deletion. By using array-based comparative genomic hybridization and fluorescent in situ hybridization to determine the boundaries of the deletions and by including data from two individuals with terminal 15q deletions but without CDH, we were able to exclude a substantial portion of the telomeric region from the genetic etiology of this disorder. Moreover, one patient with CDH harbored a small interstitial deletion. Together, these findings allowed us to define a minimal deletion region of approximately 5 Mb at chromosome 15q26.1-26.2. The region contains four known genes, of which two--NR2F2 and CHD2--are particularly intriguing gene candidates for CDH.

Abnormalities, Multiple↗
PubMed2005

Mullerian inhibiting substance suppresses tumor growth in the C3(1)T antigen transgenic mouse mammary carcinoma model.

Mullerian inhibiting substance (MIS) inhibits breast cancer cell growth in vitro. To extend the use of MIS to treat breast cancer, it is essential to test the responsiveness of mammary tumor growth to MIS in vivo. Mammary tumors arising in the C3(1) T antigen mouse model expressed the MIS type II receptor, and MIS in vitro inhibited the growth of cells derived from tumors. Administration of MIS to mice was associated with a lower number of palpable mammary tumors compared with vehicle-treated mice (P=0.048), and the mean mammary tumor weight in the MIS-treated group was significantly lower compared with the control group (P=0.029). Analysis of proliferating cell nuclear antigen (PCNA) expression and caspase-3 cleavage in tumors revealed that exposure to MIS was associated with decreased proliferation and increased apoptosis, respectively, and was not caused by a decline in T antigen expression. The effect of MIS on tumor growth was also evaluated on xenografted human breast cancer cell line MDA-MB-468, which is estrogen receptor- and retinoblastoma-negative and expresses mutant p53, and thus complements the C3(1)Tag mouse mammary tumors that do not express estrogen receptor and have functional inactivation of retinoblastoma and p53. In agreement with results observed in the transgenic mice, MIS decreased the rate of MDA-MB-468 tumor growth and the gain in mean tumor volume in severe combined immunodeficient mice compared with vehicle-treated controls (P=0.004). These results suggest that MIS can suppress the growth of mammary tumors in vivo.

Animals↗
PubMed2005

Nitrofen induces a redox-dependent apoptosis associated with increased p38 activity in P19 teratocarcinoma cells.

Nitrofen is a diphenyl ether herbicide that produces a spectrum of fetal abnormalities in rodents. To characterize the molecular mechanisms of nitrofen-mediated birth defects at the cellular level, we explored its effects on undifferentiated P19 teratocarcinoma cells. Nitrofen induces a time-dependent cell death of P19 cells that is associated with increases in TUNEL-positivity and caspase-3 cleavage suggesting that nitrofen induces P19 cell apoptosis. In addition, the increase in TUNEL-positive cells was inhibited with zVAD-fmk, suggesting that nitrofen induces a caspase-dependent apoptosis. Nitrofen treatment was associated with increased p38 MAP kinase activity, though pretreatment of cells with multiple p38 inhibitors did not affect nitrofen-mediated caspase-3 cleavage, suggesting caspase-3 cleavage is p38-independent. Nitrofen induced a dose-dependent increase in reactive oxygen species (ROS), which was accompanied by a decrease in the ratio of reduced/oxidized glutathione, indicating that nitrofen alters the cellular redox state of these cells. Furthermore, pretreatment of cells with N-acetyl cysteine gave a dose- and time-dependent reduction of caspase-3 cleavage, supporting the observations that caspase-3 cleavage is cell-redox-dependent. Therefore, nitrofen induces P19 cell apoptosis that is cell-redox-dependent and is associated with increases in p38 activity and ROS and may play a role in nitrofen-mediated birth defects.

Animals↗
PubMed2001

Mullerian inhibiting substance regulates NFkappaB signaling and growth of mammary epithelial cells in vivo.

Müllerian inhibiting substance (MIS) inhibits breast cancer cell growth in vitro through interference with cell cycle progression and induction of apoptosis, a process associated with NFkappaB activation and up-regulation of one of its important target genes, IEX-1S (Segev, D. L., Ha, T., Tran, T. T., Kenneally, M., Harkin, P., Jung, M., MacLaughlin, D. T., Donahoe, P. K., and Maheswaran, S. (2000) J. Biol. Chem. 275, 28371-28379). Here we demonstrate that MIS activates the NFkappaB signaling cascade, induces IEX-1S mRNA, and inhibits the growth of MCF10A, an immortalized human breast epithelial cell line with characteristics of normal cells. In vivo, an inverse correlation was found to exist between various stages of mammary growth and MIS type II receptor expression. Receptor mRNA significantly diminished during puberty, when the ductal system branches and invades the adipose stroma and during the expansive growth at lactation, but it was up-regulated during involution, a time of regression and apoptosis. Peripartum variations in MIS type II receptor expression correlated with NFkappaB activation and IEX-1S mRNA expression. Administration of MIS to female mice induced NFkappaB DNA binding and IEX-1S mRNA expression in the breast. Furthermore, exposure to MIS in vivo increased apoptosis in the mouse mammary ductal epithelium. Thus, MIS may function as an endogenous hormonal regulator of NFkappaB signaling and growth in the breast.

Animals↗
PubMed2001

Müllerian Inhibiting Substance lowers testosterone in luteinizing hormone-stimulated rodents.

Müllerian Inhibiting Substance (MIS) expression is inversely proportional to the serum concentration of testosterone in males after birth and in vitro studies have shown that MIS can lower testosterone production by Leydig cells. Also, mice overexpressing MIS exhibited Leydig cell hypoplasia and lower levels of serum testosterone, but it is not clear whether this is a result of MIS affecting the development of Leydig cells or their capacity to produce testosterone. To examine the hypothesis that MIS treatment will result in decreased testosterone production by mature Leydig cells in vivo, we treated luteinizing hormone (LH)-stimulated adult male rats and mice with MIS and demonstrated that it can lead to a several-fold reduction in testosterone in serum and in testicular extracts. There was also a slight decrease in 17-OH-progesterone compared to the more significant decrease in testosterone, suggesting that MIS might be regulating the lyase activity of cytochrome P450c17 hydroxylase/lyase (Cyp17), but not its hydroxylase activity. Northern analysis showed that, in both MIS-treated rats and mice, the mRNA for Cyp17, which catalyzes the committed step in androgen synthesis, was down-regulated. In rats, the mRNA for cytochrome P450 side-chain cleavage (P450scc) was also down-regulated by MIS. This was not observed in mice, indicating that there might be species-specific regulation by MIS of the enzymes involved in the testosterone biosynthetic pathway. Our results show that MIS can be used in vivo to lower testosterone production by mature rodent Leydig cells and suggest that MIS-mediated down-regulation of the expression of Cyp17, and perhaps P450scc, contributes to that effect.

17-alpha-Hydroxyprogesterone↗
PubMed2001

Tissue-engineered cells producing complex recombinant proteins inhibit ovarian cancer in vivo.

Techniques of tissue engineering and cell and molecular biology were used to create a biodegradable scaffold for transfected cells to produce complex proteins. Mullerian Inhibiting Substance (MIS) causes regression of Mullerian ducts in the mammalian embryo. MIS also causes regression in vitro of ovarian tumor cell lines and primary cells from ovarian carcinomas, which derive from Mullerian structures. In a strategy to circumvent the complicated purification protocols for MIS, Chinese hamster ovary cells transfected with the human MIS gene were seeded onto biodegradable polymers of polyglycolic acid fibers and secretion of MIS confirmed. The polymer-cell graft was implanted into the right ovarian pedicle of severe combined immunodeficient mice. Serum MIS in the mice rose to supraphysiologic levels over time. One week after implantation of the polymer-cell graft, IGROV-1 human tumors were implanted under the renal capsule of the left kidney. Growth of the IGROV-1 tumors was significantly inhibited in the animals with a polymer-cell graft of MIS-producing cells, compared with controls. This novel MIS delivery system could have broader applications for other inhibitory agents not amenable to efficient purification and provides in vivo evidence for a role of MIS in the treatment of ovarian cancer.

Animals↗
PubMed2001

Surgical treatment of congenital adrenal hyperplasia.

Unraveling of the genetics of CAH offers the possibility of earlier detection and prenatal treatment or, alternatively, blastocyst embryo selection and eventually in utero gene therapy. Endocrine, surgical, and anesthesia management after birth have improved, leading to a better outcome for these patients. In the authors' experience, early one-stage reconstructive surgery, although demanding, allows one to use all available tissue. Once mastered, the repair is actually technically easier than vaginal pull-through surgery in the adolescent. Patients go through childhood with a body image that is more concordant with normal. Neither the child nor the parents must suffer the anticipation of a major operative intervention at puberty that can cause great emotional stress and that may be more difficult. The authors have encountered situations in late adolescence in which it has been impossible to separate the urogenital sinus from below. Under these circumstances, one can consider a posterior sagittal approach in which the rectum is bivalved to allow one to approach the vagina from below in an attempt to separate it safely from the urethra and to mobilize it to the perineum. It is also feasible to consider fashioning a segment of sigmoid colon as a neovagina, realizing that mucosal drainage needs to be managed daily. The authors have also encountered the rare 46,XX patient raised as a male and committed to the male role. In these cases, the patient can be offered gonadectomy, followed by staged complex hypospadias repair, and surgery to remove Müllerian structures and, if possible, to preserve the vas, followed by prepenile scrotal repair and insertion of testicular prostheses. Children with CAH require a lifetime of care with surgical approaches that are age appropriate. These patients can lead a full and productive life. It is the physician's responsibility to make certain that these children reach their full potential with the least number of interventions, which should be designed and optimized to produce the best possible outcome.

Adolescent↗
PubMed2001

Decreased mitogen activated protein kinase activities in congenital diaphragmatic hernia-associated pulmonary hypoplasia.

BACKGROUND/PURPOSE: The mechanisms that cause pulmonary hypoplasia associated with congenital diaphragmatic hernia (CDH) currently are unknown. The authors proposed that the reduced size and immaturity of these lungs may be associated with differences in the levels of mitogen activated protein (MAP) kinase phosphorylation (extracellular signal regulated protein kinases, ERK-1 and -2). METHODS: ERK-1 activities were measured using immune-complex kinase assays on fetal whole-lung lysates obtained from both nitrofen and olive oil-treated (control) pregnant rats. In addition, ERK-1 and ERK-2 functional activities were estimated by semiquantitative Western blot analysis, using an antibody specific for the diphosphorylated (dp-ERK, activated) forms of the enzymes. RESULTS: ERK-1 activities, measured using immune-complex kinase assays, were reduced in CDH lungs compared with olive oil-treated controls (P <.02). In addition, dp-ERK-1 and dp-ERK-2 levels were found to be reduced in CDH lungs compared with controls (dp-ERK-1, P =.003; dp-ERK-2, P =.04), whereas ERK-1 and ERK-2 protein levels were unchanged. CONCLUSIONS: The lower values of ERK-1 activity and reduced amounts of dp-ERK-1 and dp-ERK-2 in lung tissue from CDH animals, suggests that ERK-1 and ERK-2 activities are reduced in pulmonary hypoplasia associated with CDH. The observed reduction in ERK-1 and ERK-2 activities implicates attenuated cell signaling upstream of the ERK-1 and -2 enzymes.

Animals↗
PubMed2001

Müllerian inhibiting substance: an instructive developmental hormone with diagnostic and possible therapeutic applications.

Dr. Alfred Jost pioneered the field of reproductive endocrinology with his seminal observation that two hormones produced by the testes are required for the male embryo to develop a normal internal reproductive tract. T induces the Wolffian ducts to differentiate into epididymides, vasa deferens, and seminal vesicles. Müllerian inhibiting substance (MIS) causes regression of the Müllerian ducts, which in its absence would normally develop into the Fallopian tubes, uterus, and upper vagina as is observed in female embryos. This review will summarize our current understanding of molecular mechanisms underlying the function of MIS both as a fetal gonadal hormone that causes Müllerian duct regression and as an adult hormone, the roles for which are currently being investigated, i.e., inhibition of steroidogenesis, germ cell development, and cancer. We will also address the regulation of MIS expression as one of the first genes expressed after the commitment of the bipotential gonads to differentiate into testes under the influence of SRY, the gene on the sex-determining region of the Y chromosome. We will discuss what is known regarding MIS signal transduction, which as with other members of the TGFbeta family of growth and differentiation factors, occurs through a heteromeric complex of single transmembrane serine/threonine kinase receptors to effect downstream signaling events, including Smad, nuclear factor-kappaB, beta-catenin, and p16 activation. Finally, we will assess the clinical relevance of studying MIS in patients with persistent Müllerian duct syndrome and our efforts to determine the therapeutic value of MIS for patients with ovarian and other MIS receptor-expressing cancers.

Aging↗
PubMed2001

SRYand architectural gene regulation: the kinetic stability of a bent protein-DNA complex can regulate its transcriptional potency.

Protein-directed DNA bending is proposed to regulate assembly of higher-order DNA-multiprotein complexes (enhanceosomes and repressosomes). Because transcriptional initiation is a nonequilibrium process, gene expression may be modulated by the lifetime of such complexes. The human testis-determining factor SRY contains a specific DNA-bending motif, the high-mobility group (HMG) box, and is thus proposed to function as an architectural factor. Here, we test the hypothesis that the kinetic stability of a bent HMG box-DNA complex can in itself modulate transcriptional potency. Our studies employ a cotransfection assay in a mammalian gonadal cell line as a model for SRY-dependent transcriptional activation. Whereas sex-reversal mutations impair SRY-dependent gene expression, an activating substitution is identified that enhances SRY's potency by 4-fold. The substitution (I13F in the HMG box; fortuitously occurring in chimpanzees) affects the motif's cantilever side chain, which inserts between base pairs to disrupt base pairing. An aromatic F13 cantilever prolongs the lifetime of the DNA complex to an extent similar to its enhanced function. By contrast, equilibrium properties (specific DNA affinity, specificity, and bending; thermodynamic stability and cellular expression) are essentially unchanged. This correlation between potency and lifetime suggests a mechanism of kinetic control. We propose that a locked DNA bend enables multiple additional rounds of transcriptional initiation per promoter. This model predicts the occurrence of a novel class of clinical variants: bent but unlocked HMG box-DNA complexes with native affinity and decreased lifetime. Aromatic DNA-intercalating agents exhibit analogous kinetic control of transcriptional elongation whereby chemotherapeutic potencies correlate with drug-DNA dissociation rates.

Amino Acid Sequence↗
PubMed2001

Müllerian inhibiting substance signaling uses a bone morphogenetic protein (BMP)-like pathway mediated by ALK2 and induces SMAD6 expression.

Signal reception of Müllerian inhibiting substance (MIS) in the mesenchyme around the embryonic Müllerian duct in the male is essential for regression of the duct. Deficiency of MIS or of the MIS type II receptor, MISRII, results in abnormal reproductive development in the male due to the maintenance of the duct. MIS is a member of the transforming growth factor-beta (TGFbeta) superfamily of secreted protein hormones that signal through receptor complexes of type I and type II serine/threonine kinase receptors. To investigate candidate MIS type I receptors, we examined reporter construct activation by MIS. The bone morphogenetic protein (BMP)-responsive Tlx2 and Xvent2 promoter-driven reporter constructs were stimulated by MIS but the TGFbeta/activin-induced p3TP-lux or CAGA-luc reporter constructs were not. The induction of Tlx2-luc was dependent upon the kinase activity of MISRII and was blocked by a dominant negative truncated ALK2 (tALK2) receptor but not by truncated forms of the other BMP type I receptors ALK1, ALK3, or ALK6. MIS induced activation of a Gal4DBD-Smad1 but not a Gal4DBD-Smad2 fusion protein. This activation could also be blocked by tALK2. The BMP-induced inhibitory Smad, Smad6, was up-regulated by MIS endogenously in Leydig cell-derived lines and is expressed in male but not female Müllerian duct mesenchyme. ALK6 has been shown to function as an MIS type I receptor. Investigation of the pattern of ALK2, MISRII, and ALK6 in the developing urogenital system demonstrated overlapping expression of ALK2 and MISRII in the mesenchyme surrounding the duct while ALK6 was observed only in the epithelium. Examination of ALK6 -/- male animals revealed no defect in duct regression. The reporter construct analysis, pattern of expression of the receptors, and analysis of ALK6-deficient animals suggest that ALK2 is the MIS type I receptor involved in Müllerian duct regression.

Activin Receptors, Type I↗
PubMed2000

Mullerian inhibiting substance inhibits ovarian cell growth through an Rb-independent mechanism.

Müllerian inhibiting substance (MIS), a transforming growth factor-beta family member, causes regression of the Müllerian duct in male embryos. MIS overexpression in transgenic mice ablates the ovary, and MIS inhibits the growth of ovarian cancer cell lines in vitro, suggesting a key role for this hormone in postnatal development of the ovary. This report describes a mechanism for MIS-mediated growth inhibition in both a human epithelial ovarian cancer cell line and a cell line derived from normal ovarian surface epithelium, which is the origin of human epithelial ovarian cancers. MIS-treated cells accumulated in the G(1) phase of the cell cycle and subsequently underwent apoptosis. MIS up-regulated the cyclin-dependent kinase inhibitor p16 through an MIS type II receptor-mediated mechanism and inhibited growth in the absence of detectable or inactive Rb protein. Prolonged treatment with MIS down-regulated the Rb-related protein p130 and increased the Rb family-regulated transcription factor E2F1, overexpression of which inhibited growth. These findings demonstrate that p16 is required for MIS-mediated growth inhibition in ovarian epithelial cells and tumor cells and suggest that up-regulation of E2F1 also plays a role in this process.

Animals↗
PubMed2000

Mullerian inhibiting substance inhibits breast cancer cell growth through an NFkappa B-mediated pathway.

Müllerian inhibiting substance (MIS), a member of the transforming growth factor-beta superfamily, induces regression of the Müllerian duct in male embryos. In this report, we demonstrate MIS type II receptor expression in normal breast tissue and in human breast cancer cell lines, breast fibroadenoma, and ductal adenocarcinomas. MIS inhibited the growth of both estrogen receptor (ER)-positive T47D and ER-negative MDA-MB-231 breast cancer cell lines, suggesting a broader range of target tissues for MIS action. Inhibition of growth was manifested by an increase in the fraction of cells in the G(1) phase of the cell cycle and induction of apoptosis. Treatment of breast cancer cells with MIS activated the NFkappaB pathway and selectively up-regulated the immediate early gene IEX-1S, which, when overexpressed, inhibited breast cancer cell growth. Dominant negative IkappaBalpha expression ablated both MIS-mediated induction of IEX-1S and inhibition of growth, indicating that activation of the NFkappaB signaling pathway was required for these processes. These results identify the NFkappaB-mediated signaling pathway and a target gene for MIS action and suggest a putative role for the MIS ligand and its downstream interactors in the treatment of ER-positive as well as negative breast cancers.

Animals↗
PubMed2000

Activin receptor-like kinase 1 modulates transforming growth factor-beta 1 signaling in the regulation of angiogenesis.

The activin receptor-like kinase 1 (ALK1) is a type I receptor for transforming growth factor-beta (TGF-beta) family proteins. Expression of ALK1 in blood vessels and mutations of the ALK1 gene in human type II hereditary hemorrhagic telangiectasia patients suggest that ALK1 may have an important role during vascular development. To define the function of ALK1 during development, we inactivated the ALK1 gene in mice by gene targeting. The ALK1 homozygous embryos die at midgestation, exhibiting severe vascular abnormalities characterized by excessive fusion of capillary plexes into cavernous vessels and hyperdilation of large vessels. These vascular defects are associated with enhanced expression of angiogenic factors and proteases and are characterized by deficient differentiation and recruitment of vascular smooth muscle cells. The blood vessel defects in ALK1-deficient mice are reminiscent of mice lacking TGF-beta1, TGF-beta type II receptor (TbetaR-II), or endoglin, suggesting that ALK1 may mediate TGF-beta1 signal in endothelial cells. Consistent with this hypothesis, we demonstrate that ALK1 in endothelial cells binds to TGF-beta1 and TbetaR-II. Furthermore, the ALK1 signaling pathway can inhibit TGF-beta1-dependent transcriptional activation mediated by the known TGF-beta1 type I receptor, ALK5. Taken together, our results suggest that the balance between the ALK1 and ALK5 signaling pathways in endothelial cells plays a crucial role in determining vascular endothelial properties during angiogenesis.

Activin Receptors↗
PubMed2000

Endogenous expression of Müllerian inhibiting substance in early postnatal rat sertoli cells requires multiple steroidogenic factor-1 and GATA-4-binding sites.

Müllerian inhibiting substance (MIS) is a key element required to complete mammalian male sex differentiation. The expression pattern of MIS is tightly regulated in fetal, neonatal, and prepubertal testes and adult ovaries and is well conserved among mammalian species. Although several factors have been shown to be essential to MIS expression, its regulatory mechanisms are not fully understood. We have examined MIS promoter activity in 2-day postnatal primary cultures of rat Sertoli cells that continue to express endogenous MIS mRNA. Using this system, we found that the region between human MIS-269 and -192 is necessary for full MIS promoter activity. We identified by DNase I footprint and electrophoretic mobility-shift analyses a distal steroidogenic factor-1 (SF-1)-binding site that is essential for full promoter activity. Mutational analysis of this new distal SF-1 site and the previously identified proximal SF-1 site showed that both are necessary for transcriptional activation. Moreover, the proximal promoter also contains multiple GATA-4-binding sites that are essential for functional promoter activity. Thus multiple SF-1- and GATA-4-binding sites in the MIS promoter are required for normal tissue-specific and developmental expression of MIS.

Adenoviridae↗
PubMed2000

Human Müllerian-inhibiting substance promoter contains a functional TFII-I-binding initiator.

Müllerian-inhibiting substance (MIS) plays an essential role in mammalian male sexual development; thus, it is important to determine how the tightly regulated expression of the MIS gene is transcriptionally controlled. Transcription of eukaryotic genes is dependent on regulatory elements in the enhancer and one or both distinct elements in the core promoter: the TATA box, and the initiator (Inr) element. Because the human MIS gene does not contain a consensus TATA and has not been reported to contain an Inr element, we hypothesized that the initiator region of the core promoter was essential for promoter activity. Transient transfection assays were conducted using an immortalized Embryonic Day 14.5 male rat urogenital ridge cell line (CH34) that expresses low levels of MIS. These studies revealed that promoter activity is dependent on the region around the start site (-6 to +10) but not on the nonconsensus TATA region. Electrophoretic mobility shift assays demonstrated that the human MIS initiator sequence forms a specific DNA-protein complex with CH34 cell nuclear extract, HeLa cell nuclear extract, and purified TFII-I. This complex could be blocked or supershifted by the addition of antibodies directed against TFII-I. These data suggest that the human MIS gene contains a functional initiator that is specifically recognized by TFII-I.

Animals↗