MutT Homolog 1 (MTH1) maintains multiple KRAS-driven pro-malignant pathways.

Oncogenic RAS promotes production of reactive oxygen species (ROS), which mediate pro-malignant signaling but can also trigger DNA damage-induced tumor suppression. Thus RAS-driven tumor cells require redox-protective mechanisms to mitigate the damaging aspects of ROS. Here, we show that MutT Homolog 1 (MTH1), the mammalian 8-oxodGTPase that sanitizes oxidative damage in the nucleotide pool, is important for maintaining several KRAS-driven pro-malignant traits in a nonsmall cell lung carcinoma (NSCLC) model. MTH1 suppression in KRAS-mutant NSCLC cells impairs proliferation and xenograft tumor formation. Furthermore, MTH1 levels modulate KRAS-induced transformation of immortalized lung epithelial cells. MTH1 expression is upregulated by oncogenic KRAS and correlates positively with high KRAS levels in NSCLC human tumors. At a molecular level, in p53-competent KRAS-mutant cells, MTH1 loss provokes DNA damage and induction of oncogene-induced senescence. In p53-nonfunctional KRAS-mutant cells, MTH1 suppression does not produce DNA damage but reduces proliferation and leads to an adaptive decrease in KRAS expression levels. Thus, MTH1 not only enables evasion of oxidative DNA damage and its consequences, but can also function as a molecular rheostat for maintaining oncogene expression at optimal levels. Accordingly, our results indicate MTH1 is a novel and critical component of oncogenic KRAS-associated malignancy and its inhibition is likely to yield significant tumor-suppressive outcomes in KRAS-driven tumors.

A novel nuclear role for the Vav3 nucleotide exchange factor in androgen receptor coactivation in prostate cancer.

Increased androgen receptor (AR) transcriptional activity mediated by coactivator proteins may drive castration-resistant prostate cancer (CRPC) growth. Vav3, a Rho GTPase guanine nucleotide exchange factor (GEF), is overexpressed in human prostate cancers, particularly in models of CRPC progression. Vav3 coactivates AR in a Vav3 pleckstrin homology (PH) domain-dependent but GEF-independent manner. Ectopic expression of Vav3 in androgen-dependent human prostate cancer cells conferred robust castration-resistant xenograft tumor growth. Vav3 but not a Vav3 PH mutant greatly stimulated interaction between the AR amino and carboxyl termini (N-C interaction), which is required for maximal receptor transcriptional activity. Vav3 was distributed between the cytoplasm and nucleus with nuclear localization-dependent on the Vav3 PH domain. Membrane targeting of Vav3 abolished Vav3 potentiation of AR activity, whereas nuclear targeting of a Vav3 PH mutant rescued AR coactivation, suggesting that nuclear localization is an important function of the Vav3 PH domain. A nuclear role for Vav3 was further demonstrated by sequential chromatin immunoprecipitation assays, which revealed that Vav3 and AR were recruited to the same transcriptional complexes of an AR target gene enhancer. These data demonstrate the importance of Vav3 in CRPC and define a novel nuclear function of Vav3 in regulating AR activity.

Cooperative actions of hepatocyte growth factor and 1,25-dihydroxyvitamin D3 in osteoblastic differentiation of human vertebral bone marrow stromal cells.

Bone formation and remodeling require continuous generation of osteoprogenitor cells from bone marrow stromal cells (MSC), which generate and respond to a variety of growth factors with putative roles in hematopoiesis and mesenchymal differentiation. In this study we examine the interaction of two such factors on the maturation of skeletal components. We previously reported that these factors, hepatocyte growth factor (HGF) and 1,25-dihydroxyvitamin D(3) (vitD(3)), act together to increase alkaline phosphatase in chondroblasts. We now describe the cooperative effect of these agents on MSC isolated and cultured from human vertebral bone marrow. MSC (passages 3-9) isolated from bone marrow cells of human vertebrae (T1-L5) from 22-36-year-old normal donors were first expanded in vitro and then plated in the presence or absence of 10 ng/mL HGF and/or 10 nmol/L vitD(3), for 7-18 days. HGF treatment increased cell proliferation 2.5-fold, with no effect on alkaline phosphatase activity. Whereas vitD(3) treatment inhibited cell growth by 50%, alkaline phosphatase activity was stimulated eightfold, although no mineralization was observed. HGF together with vitD(3) increased cell proliferation 1.5-fold and alkaline phosphatase activity 13-fold over untreated control. Moreover, mineralization was detected only with this combination. Our findings provide evidence that HGF in concert with vitamin D may promote growth and differentiation of human MSC into osteogenic cells.

Gap-junctional communication is required for the maturation process of osteoblastic cells in culture.

Osteoblastic cells in long-term culture undergo a phenotypic maturation process leading to extracellular matrix (ECM) production and bone nodule (BN) formation. Cell-to-cell communication via gap junctions (GJC) can be detected between osteoblastic cells within 24 h of plating. We evaluated, in long-term cultures of osteoblastic cells, the effect of inhibiting GJC on the phenotypic maturation process and the expression of specific genes associated with this process. MC3T3-E1 cells were plated, and, after 24 h (day 0), cells were exposed to 18-alpha-glycyrrhetinic acid (AGA), a nontoxic reversible inhibitor of GJC. GJC, alkaline phosphatase (AP) activity, BN formation, and the relative level of transcripts encoding osteocalcin (OC), bone sialoprotein (bSP), osteopontin (OP), collagen alpha1 type I (alpha1ICol), and elongation factor-1a (EF1a) were evaluated on day 0 and every 4-7 days thereafter through day 30. GJC was assessed by fluorescent dye transfer. Gene expression was analyzed by northern blot and semiquantitative reverse transcription-polymerase chain reaction. GJC was detectable at day 0 and increased with time in culture. AGA (100 micromol/L) strongly inhibited GJC at all timepoints tested. Moreover, AGA-exposed cells showed a dose-dependent decrease in AP activity and a delay in the appearance of BN. This delayed phenotypic expression coincided with an inhibitory effect on the expression of the osteoblast-specific genes OC and bSP. Expression of alpha1ICol mRNA was also affected, but to a lesser extent, whereas OP and EF1a were not affected. Similar results were obtained with oleamide, an additional reversible inhibitor of GJC. In contrast, cells exposed to either vehicle or 100 micromol/L glycyrrhizic acid (a noninhibitory glycoside of 18-beta-glycyrrhetinic acid) were indistinguishable from untreated cells for all parameters evaluated. We conclude that GJC inhibition interferes with the maturation process of osteoblastic cells in culture, possibly by affecting signals regulating the expression of genes involved in the maturation/differentiation of the osteoblastic phenotype.

Gap-junctional communication mediates parathyroid hormone stimulation of mineralization in osteoblastic cultures.

Previously we showed that physiological levels of parathyroid hormone (PTH) can increase the mineralization of extracellular matrix (ECM) by osteoblast-like cells in vitro. In this study, we assess the role of gap-junctional intercellular communication (GJC) in the PTH-enhanced mineralization of ECM in MC3T3-E1 cells, a murine culture model of osteoblastic differentiation. Messenger RNA and protein for connexin 43 (Cx43), the major component of MC3T3-E1 gap junctions, and GJC increased as the cells progressed toward a mature phenotype. Immunocytochemistry showed accumulation of Cx43 at the area of close contact between cells. The timing of the PTH treatment that increased matrix mineralization in these cells coincided with the highest expression of Cx43 and GJC. Administration of 18-alpha-glycyrrhetinic acid (AGA) promptly blocked GJC in cultures of MC3T3-E1 cells in a dose-dependent and reversible manner at all times tested during the culture period. Treatment with AGA, but not with an inactive analog, reversed the PTH-induced ECM mineralization. These data suggest that GJC mediates anabolic actions of PTH related to osteoblast-mediated mineralization.

Testes exhibit elevated expression of calcitonin gene-related peptide receptor component protein.

Calcitonin gene-related peptide (CGRP) receptor component protein (RCP) is a novel protein that modulates CGRP responsiveness in a variety of cell types. Using probes based on the isolation of CGRP-RCP complementary DNA (cDNA) from a guinea pig organ of Corti cDNA library, we cloned human (h) and mouse (m) CGRP-RCP cDNAs, both of which encode 148-residue proteins that at the amino acid levels are approximately 88% identical to each other and to the 146-residue guinea pig CGRP-RCP. Northern blot analysis confirmed the presence of CGRP-RCP messenger RNA in all of the human and mouse tissues tested. In these human tissues, hCGRP-RCP messenger RNA (major band at approximately 3.1 kb, minor band at approximately 7.5 kb) was most prevalent in the testis. In the mouse, the highest abundance of CGRP-RCP RNA was clearly in the testis (major band at approximately 1.6 kb, minor band at approximately 1.1 kb). Based on this tissue distribution of RNA, we sought to identify the cells in the murine testis that contained CGRP-RCP protein. Numerous antisera generated against hCGRP-RCP, including one to recombinant hCGRP-RCP, exhibited strong immunoreactivity localized to the head region of spermatozoa. No CGRP-RCP immunoreactivity was observed in other cells at less mature stages of sperm maturation, in Sertoli or interstitial (Leydig) cells, or in human spermatozoa. Murine epididymal (mature) spermatozoa exhibited CGRP-RCP immunoreactivity identical to that of testicular spermatozoa. Spermatozoa that underwent an experimentally induced acrosome reaction (acrosomal discharge) lost their CGRP-RCP immunoreactivity. Therefore, it appears that CGRP-RCP is associated with the acrosome, suggesting that it may play an important role in reproduction.

Expression of rat thyrotropin-releasing hormone (TRH) gene in TRH-producing tissues of transgenic mice requires sequences located in exon 1.

TRH, an amidated tripeptide secreted by certain hypothalamic neurons, is a principal regulator of TSH secretion and thyroid hormone release. TRH is also produced by other neurons in the central nervous system, where it appears to function as a neuromodulator or neurotransmitter, and by certain endocrine cells, where it may act as an autocrine or paracrine factor. The genomic organization of the rat TRH (rTRH) gene is well understood; however, the domains of the rTRH gene that regulate expression are less well characterized. We observed that the region between -47 and +6 of the rTRH gene (relative to the transcription start site at +1) was active in CA-77 cells, a medullary thyroid carcinoma cell line model of TRH production, but was not active in transgenic mice. Inclusion of most of exon 1 (84 out of 103 bp; -47 to +84) increased promoter activity in CA-77 cells and was active in transgenic mice, principally in tissues that normally express the TRH gene. Further lengthening of the 5' end to -243, -547, or -776 retained this expression in TRH-producing tissues in transgenic mice, while further increasing activity in CA-77 cells. These results suggest that cis element(s) located within exon 1 are necessary for the expression of the rTRH gene in vivo.

Calcitonin gene-related peptide (CGRP) in the developing mouse limb.

The mitogenic effects of neuropeptides and their localization to developing tissues suggest an important role for these peptides during gestation. We examined the expression and action of the neuropeptide calcitonin gene-related peptide (CGRP) in the developing mouse limb bud, an excellent model system for studying musculoskeletal development. CGRP immunoreactivity (CGRP-ir) was detected in the developing limb at day 16.5 of gestation (E 16.5) and was limited to nerve fibers surrounding blood vessels, within the developing muscle or in close proximity to the developing cartilaginous skeleton. Although CGRP-ir was not observed until E 16.5, limb buds were responsive to CGRP as early as E 11.5. Within 5 min of exposure to CGRP (10(-8) to 10(-7) M) a 2--3-fold increase in cyclic AMP (cAMP) levels was observed. This CGRP-induced increase in cAMP was abolished by the addition of human CGRP8-37, a CGRP receptor antagonist. This result suggests that the effect on cAMP was mediated by the interaction of CGRP with CGRP receptors. Our findings indicate that mouse limbs are responsive to CGRP when they are composed of primarily undifferentiated mesenchyme and that CGRP-ir appears at a later stage of development in association with cartilage and muscle differentiation.

Neuroendocrine peptides stimulate adenyl cyclase in normal and malignant prostate cells.

Elevations of intracellular cAMP in human prostate cancer cells have been shown to increase invasiveness and to promote neuronal differentiation. Since neuroendocrine peptides capable of activating adenyl cyclase are present in prostatic nerves and epithelial neuroendocrine cells, we investigated normal and malignant human prostate cells for changes in intracellular cAMP in response to the prostatic peptides vasoactive intestinal peptide (VIP), calcitonin (CT), and calcitonin gene-related peptide (CGRP). Normal prostate epithelial cells and LNCaP prostate cancer cells exhibited, respectively, 6- and 30-fold increases in intracellular cAMP in response to VIP. ALVA-31 and PPC-1 prostate cancer cells demonstrated 20- to 200-fold increases in cAMP in response to CGRP, while normal epithelial cells and LNCaP cells exhibited smaller (2- to 6-fold) responses. Only DU-145 cells increased cAMP substantially in response to CT. VIP receptor mRNA was identified by Northern blot analysis only in those cells that responded to VIP. CT receptor mRNA was identified only in DU-145 cells by polymerase chain reaction and Southern blot analysis. These results suggest that VIP and possibly CGRP receptors are likely to be present in both normal and malignant prostate cells. VIP or CGRP may regulate secretion of proteases by normal or prostate cancer cells and may influence epithelial cell differentiation.

Transgenic mice expressing a constitutively active retinoic acid receptor in the lens exhibit ocular defects.

Retinoic acid receptors (RARs) modulate gene expression following association with retinoic acid (RA). In transient transfection, an RAR alpha-beta-galactosidase fusion protein (RAR-LacZ) was able to transactivate expression in the absence of RA. When expressed in the ocular lens of transgenic mice, this constitutively active RAR-LacZ fusion gene resulted in founder and progeny animals that exhibited cataracts and microphthalmia, both being characteristics of retinoid-induced teratogenesis. The transgenic phenotypes indicate that retinoid teratogenesis can be mimicked by expression of a constitutively active RAR-LacZ fusion protein in retinoid-sensitive tissues.

Transgenic indicator mice for studying activated retinoic acid receptors during development.

Retinoic acid (RA) receptors (RARs) are ligand-inducible transcription factors that bind to specific DNA sequences associated with the regulatory regions of RA-regulatable genes. Since RA has been implicated as an important factor both in normal development and in teratological studies, one would like to have a model system that detects the presence of activated receptors during development. We have constructed a recombinant reporter gene that has three copies of the RA response element (RARE) from the RAR beta-2 promoter 5' to the herpes simplex virus thymidine kinase promoter; this regulatory region is coupled to the bacterial beta-galactosidase reporter gene. This construct was RA inducible in transient transfection assays in F9 embryonal carcinoma cells. Transgenic embryos with this reporter gene construct exhibited restricted and reproducible patterns of beta-galactosidase activity during embryogenesis, beginning between gestational ages day 7.5 and 8.5. At day 8.5, beta-galactosidase activity was detected in the closed neurotube and somites. Day 8.5 embryos, from pregnant females fed RA 14 hr earlier, exhibited a greater intensity and distribution of beta-galactosidase activity. Similarly, at later stages of gestation, maternal RA exposure resulted in enhanced embryonic beta-galactosidase expression. This type of transgenic indicator mouse should be useful in detailing the role of activated RARs during embryonic development.

Role of the mouse visceral yolk sac in nutrition: inhibition by a somatomedin inhibitor.

A low molecular weight somatomedin inhibitory serum fraction (SI), obtained from streptozotocin-induced diabetic rats, causes morphological abnormalities and growth reduction in mouse embryos grown in whole embryo culture (WEC). These abnormalities are thought to be caused, at least in part, by a failure of the visceral yolk sac (VYS) to properly degrade proteins, a process that normally provides the conceptus with amino acids and peptides for de novo protein synthesis (histiotrophic nutrition). To test this hypothesis, embryos exposed to the SI were provided with a mixture of ten essential amino acids (supplemented group) in an attempt to circumvent SI-induced VYS dysfunction. Results showed that 82.4% (14/17) of embryos in the amino acid-supplemented group exhibited improved growth and development compared to those embryos exposed to medium containing the SI alone (unsupplemented group). Supplemented embryos showed greater expansion of the brain regions, improved visceral arch development, and increased protein content compared to nonsupplemented SI-treated embryos. However, these parameters were still reduced compared to controls. VYSs from both the unsupplemented and amino acid-supplemented groups were identical with respect to alterations in morphology and increased protein content compared to VYSs from conceptuses cultured in control medium (with or without amino acid supplementation). The improvement in embryonic growth and development due to amino acid supplementation in spite of VYS abnormalities supports the hypothesis that nutritional deprivation is one aspect of SI-induced teratogenesis.

Effects of maternal diabetes on embryogenesis.

Through the use of the in vitro technique of mammalian whole embryo culture, the factors and mechanisms responsible for the increased incidence of congenital malformations among infants of diabetic mothers have been investigated. During early stages of embryogenesis, serum from streptozotocin-induced diabetic rats, hyperglycemia, hypoglycemia, hyperketonemia (beta-hydroxybutyrate), and low molecular weight somatomedin inhibitors are teratogenic or growth inhibitory, or both. Furthermore, combinations of these factors interact to increase the risk of a malformation occurring. In contrast, other factors, such as hyperinsulinemia and excess leucine, palmitic acid, and acetoacetate, have little or no teratogenic potential. Mechanisms of action of the factors are varied and may include alterations of arachidonic acid metabolism (hyperglycemia), glycolysis (hypoglycemia), DNA synthesis (beta-hydroxybutyrate), and embryonic nutrition (somatomedin inhibitors). The results demonstrate that the origin of the diabetic embryopathy is multifactorial, that many of the congenital defects are induced early in gestation before the diabetic woman may realize she is pregnant, and that insulin therapy reduces the risk.

Somatomedin inhibitors from human serum produce abnormalities in mouse embryos in culture.

Two human serum fractions, one from normal individuals (Mr 1,150-1,310 daltons) and the other (Mr 800-1,100 daltons) from patients suffering with uremia (renal failure, azotemia), were added to the medium used to grow embryos in whole-embryo culture (WEC) beginning at the 3-5 (day 9) or 18-21 (day 10) somite stage. Both of these fractions possessed somatomedin (insulin-like growth factor) inhibitory activity. Day 9 embryos exposed to either of the serum fractions for 24 hr exhibited incomplete rotation and neural tube closure defects and were smaller than control embryos (decreased total protein content). Developmental abnormalities induced in day 10 embryos following 24 hr in culture included a marked decrease in expansion of the brain regions, hypoplasia of the first two branchial arches, and decreased amounts of total protein compared to controls. The visceral yolk sacs (VYSs) of somatomedin inhibitor (SI)-exposed conceptuses were opaque, and those from day 10 conceptuses contained significantly more protein than controls. Morphologically, the VYS endoderm cells from SI-exposed embryos contained a much higher density of "vacuoles" than controls. These results mimic those produced by exposure of conceptuses to an SI of Mr800-1,100 obtained from the serum of diabetic rats and suggest that similar substances and mechanisms are involved.

Improved growth and development of presomite mouse embryos in whole embryo culture.

A rotator whole embryo culture system was used to assess the growth and development of late-primitive-streak-stage (Theiler stage 9-10) mouse embryos to the limb-bud stage of organogenesis in a variety of media containing combinations of mouse serum (MS), rat serum (RS), and Tyrode's buffer (TB). The results demonstrate that embryonic growth and morphogenesis to the early limb-bud stage (20 somite pairs; 48-h total culture period) mimicked that in vivo when embryos were grown for 24 h in combinations of MS:RS:TB 1:2:1 or 2:1:1 (v/v/v) and then were transferred to fresh medium containing RS:TB 3:1 at the early somite stage. When the culture period was extended for an additional 24 h (total 72-h culture period) embryonic growth retardation was observed. Regardless of the medium employed, superior growth was observed in embryos transferred at the early somite stage when compared to embryos cultured continuously in the same medium for the entire 48- or 72-h culture period.

Potential role of somatomedin inhibitors in the production of diabetic embryopathies.

Mouse conceptuses at the 18-21-somite stage were grown for 2-24 h in vitro in the presence of a serum fraction (Mr = 800-1,080 daltons) possessing somatomedin-inhibitory activity (SI) isolated from diabetic rats. Following an 8-h exposure to the SI, DNA and incorporation of 3H-thymidine were reduced in the embryos while 12 h was required to observe a reduction in total protein and RNA. At the 24-h time point, the neurectoderm was thinner than in controls, and autoradiograms of this region showed a substantial decrease in grain density with 3H-thymidine, but not 3H-leucine or -uridine. Effects of the visceral yolk sac (VYS) preceded those on the embryo. The cytoplasm of the VYS endoderm cells from conceptuses exposed to the SI contained many vacuoles by 4 h, which were larger by 24 h. Total protein was greater than in controls from 4 h onward, although 3H-leucine incorporation, which had increased after 2 h of SI exposure, returned to control levels by 8 h. As seen by SDS-polyacrylamide gel electrophoresis, VYSs from conceptuses exposed to the SI for 4 or 24 h were enriched (compared to control VYSs) in four protein bands also present in the culture medium (primarily rat serum), suggesting that protein degradation and/or transfer of amino acids and peptides to the embryo was inhibited in these VYSs. Such a conclusion was supported by a quantitative decrease in proteins and amino acids in the exocoelomic fluid of conceptuses exposed to the SI for 24 h. The altered processing of proteins may therefore represent a primary cause of the SI-induced embryonic abnormalities.

Somatomedin inhibitors from diabetic rat serum alter growth and development of mouse embryos in culture.

Offspring of diabetic humans and laboratory animals have been shown to have a higher incidence of congenital malformations with attendant growth retardation. These defects have been attributed to alterations in the intrauterine environment and specifically to changes in maternal serum factors, e.g., glucose and ketone bodies. Our investigation examines the potential teratogenicity of a low-molecular-weight (940) serum fraction with demonstrated somatomedin inhibitory activity isolated by column chromatography from streptozocin-induced diabetic rats. Mouse embryos were exposed to control or the inhibitor fraction at concentrations of 0.25-0.6% vol/vol (0.9-3.0 micrograms protein/ml culture medium) in whole embryo culture and evaluated for the presence of malformations and growth retardation. Embryos exposed to inhibitor during the period of neurulation (3-5 somites) exhibited neural tube and craniofacial defects, whereas those exposed during early limb bud stages (18-19 somites) exhibited abnormalities of the forebrain and face. In addition, both stages were growth retarded. Control fractions produced no abnormalities. These results demonstrate a potential role for somatomedin inhibitors in diabetic embryopathy and suggest that factors other than hyperglycemia and hyperketonemia may contribute to the higher incidence of malformations among infants of diabetic mothers.

Segregation of chromosomes into the spermatozoa of a man heterozygous for a 14;21 Robertsonian translocation.

Twenty-four spermatozoa from a man heterozygous for a Robertsonian translocation (45,XY,-14,-21,+t(14q;21q) were studied cytogenetically in order to determine the meiotic segregation of the translocation. When compared to the expected 1:1 ratio we observed a greater number of chromosomally normal sperm than sperm with the balanced translocation. Three sperm carried the translocation in an unbalanced form.

Sperm chromosome analysis of a man heterozygous for a pericentric inversion of chromosome 3.

Using a procedure in which human sperm were allowed to fertilize zona-free golden hamster (Mesocricetus auratus) eggs in vitro, the sperm chromosomes of a man heterozygous for inv(3) (p11q11) were analyzed. When the chromosomes were Q-banded, the inverted chromosome had the bright centromeric band on the short arm rather than on the long arm, as was seen in the normal No. 3. One hundred and eleven sperm chromosome spreads were examined, of which 64 contained the normal chromosome and 47 the inverted one. This was not significantly different from the expected 1:1 ratio. No sperm containing a chromosome imbalance caused by a crossover within the inversion were seen. Ten (8.1%) of the sperm contained chromosome abnormalities unrelated to the inversion. The ratio of X- to Y-bearing sperm was 55:45.

Cytogenetic analysis of Q-banded pronuclear chromosomes in fertilized Syrian hamster eggs.

The frequency and type of chromosome abnormalities were analyzed in 917 female pronuclei in Syrian hamster eggs fertilized by human sperm. Analysis at this stage allows detection of errors which have occurred during meiosis I and II. The chromosomes were Q-banded to identify individual chromosomes and detect subtle alterations. Thirty-three (3.6%) of the hamster egg complements were abnormal: 19 (2.1%) were hypohaploid, seven (0.76%) were hyperhaploid, two (0.2%) had double aneuploidy, and five (0.5%) had a structural chromosome abnormality. Since there were significantly more hypohaploid than hyperhaploid complements, a conservative estimate of aneuploidy can be derived by doubling the frequency of hyperhaploid complements. Thus a minimal estimate of aneuploidy (single, 1.5%, and double, 0.2%) is 1.7% and a minimal estimate of the total frequency of abnormalities is 2.2%. All chromosome groups were represented among the aneuploid complements suggesting that all chromosomes are susceptible to non-disjunction.