Knockdown of RARB2 identifies a dual role in cancer.

Two chemoprevention trials have shown that retinoic acid (RA) may be harmful in patients at risk for lung cancer, and RA administration to this high-risk group results in RARB2 reactivation. Although RARB2 is thought to possess tumor suppressive activity, its expression has recently been correlated with poorer prognosis in patients with nonsmall cell lung cancer. We hypothesized that RARB2 expression is necessary for the growth and maintenance of the oncogenic phenotype in lung cancer cells in which RARB2 has not been inactivated. We tested various antisense oligodeoxynucleotides (ASO) against RARB2 in multiple lung cancer cell lines and used microarray technology to compare the patterns of gene expression following ASO treatment versus RA treatment in the A-549 lung cancer cell line. We show that ASO treatment reduces proliferation and causes apoptosis in 3 RARB2-expressing lung cancer cell lines but has no apparent effect in at least two other lung cancer cells lines having lost RARB2 expression or one normal lung RARB2-expressing cell line; we demonstrate a correlation between resulting RARB2 expression levels and cell growth; and identify transcriptional effects related to both RA and RARB2 signaling. In particular, five genes known to contribute to carcinogenesis or chemotherapeutic resistance are down-regulated following ASO treatment: three of these are up-regulated following RA treatment. This work demonstrates a dual role for RARB2 (tumor suppression and tumor promotion) and identifies a challenge with respect to using RARB2 as a target for treatment or prevention strategies.

A modified protocol for bisulfite genomic sequencing of difficult samples.

The bisulfite genomic sequencing protocol is a widely used method for analyzing DNA methylation. It relies on the deamination of unmethylated cytosine residues to uracil; however, its high rates of DNA degradation and incomplete cytosine to uracil conversion often lead to failed experiments, uninformative results, and false positives. Here, we report the addition of a single-step multiple restriction enzyme digestion (MRED) designed to differentially digest polymerase chain reaction products amplified from unconverted DNA while leaving those of converted DNA intact. We show that for our model system, RARB2 P2 promoter, use of MRED increased informative sequencings ninefold, and MRED did not alter the clonal representation in one fully methylated cell line, H-596, treated or not with 5-azadeoxycytidine, a methylation inhibitor. We believe that this method may easily be adapted for analyzing other genes and provide guidelines for selecting the most appropriate MRED restriction enzymes.

Allelic methylation bias of the RARB2 tumor suppressor gene promoter in cancer.

Retinoic acid receptor B2 (RARB2) is frequently inactivated in cancer. Methylation in the 5'-untranslated region and first exon is known to play a role; however, few studies have analyzed the detailed methylation pattern of the promoter region. We show that hypo- and hypermethylated alleles coexist in 5/11 cell lines in which RARB2 is inactivated. We present evidence supporting the mitotic transmission of these divergent methylation patterns and find a correlation between methylation divergence and heterozygosity at the 3p24 loci, suggesting an allelic methylation bias in these lines. Using a newly devised strategy based on allelic identification via methylation-sensitive restriction enzyme digestion combined with the use of a single nucleotide polymorphism, rs755661, we demonstrate that such a bias exists in three cancer cell specimens heterozygous at rs755661 and therefore amenable to this study. This previously unreported phenomenon of allelic methylation bias suggests that a promoter methylation-independent mechanism may be responsible for inactivation at the hypomethylated allele and this inactivation is reminiscent of an aberrant form of de novo imprinting. Approaches to interpreting methylation data should incorporate the notion of allelic methylation bias.

Genome-wide association study for Crohn's disease in the Quebec Founder Population identifies multiple validated disease loci.

Genome-wide association (GWA) studies offer a powerful unbiased method for the identification of multiple susceptibility genes for complex diseases. Here we report the results of a GWA study for Crohn's disease (CD) using family trios from the Quebec Founder Population (QFP). Haplotype-based association analyses identified multiple regions associated with the disease that met the criteria for genome-wide significance, with many containing a gene whose function appears relevant to CD. A proportion of these were replicated in two independent German Caucasian samples, including the established CD loci NOD2 and IBD5. The recently described IL23R locus was also identified and replicated. For this region, multiple individuals with all major haplotypes in the QFP were sequenced and extensive fine mapping performed to identify risk and protective alleles. Several additional loci, including a region on 3p21 containing several plausible candidate genes, a region near JAKMIP1 on 4p16.1, and two larger regions on chromosome 17 were replicated. Together with previously published loci, the spectrum of CD genes identified to date involves biochemical networks that affect epithelial defense mechanisms, innate and adaptive immune response, and the repair or remodeling of tissue.

Enalaprilat blunts reflexive increases in cardiac interstitial norepinephrine in conscious rats.

OBJECTIVE: We have reported that acute administration of enalaprilat, an angiotensin converting enzyme inhibitor, induces less reflexive increase in lumbar sympathetic nerve activity in spontaneously hypertensive rats (SHRs) than nicardipine, a dihydropyridine calcium-channel blocker. The current study was conducted to determine if angiotensin converting enzyme inhibitors likewise suppress cardiac sympathetic activation. DESIGN: Cardiac interstitial levels of norepinephrine were measured in fully conscious SHRs before and after acute blood pressure lowering with enalaprilat or nicardipine. METHODS: Microdialysis probes were inserted into the left ventricular wall of SHRs. Twenty-four to 48 hours post-implantation, myocardial interstitial fluid was collected in fully conscious rats during a 60-min baseline period. Mean arterial pressure was lowered 20 mmHg with intravenous infusion of enalaprilat or nicardipine. During continuous enalaprilat or nicardipine infusion, myocardial interstitial fluid was again collected. Norepinephrine levels were assayed in the perfusate. CONCLUSIONS: Enalaprilat-induced reduction in mean arterial pressure did not significantly increase cardiac interstitial norepinephrine levels. In contrast, nicardipine-induced reduction in blood pressure was associated with a significant increase in interstitial norepinephrine levels. These results indicate that enalaprilat suppresses reflexive sympathetic activation of the heart during acute blood pressure lowering. These results may be clinically relevant in that reductions in end-organ sympathetic stimulation may enhance the long-term cardiovascular benefit of angiotensin converting enzyme inhibitors.

Angiotensin II modulates catecholamine release into interstitial fluid of canine myocardium in vivo.

This study tested the hypothesis that exogenous infusion of angiotensin II (ANG II) leads to the release of catecholamines [norepinephrine (NE) and epinephrine (EPI)] into the cardiac interstitial fluid (ISF) space of dogs with adrenals intact (AI) (n = 7) and with adrenals clamped (AC) (n = 5). LV ISF samples were collected at 3-min intervals during administration of ANG II (100 microM ANG II at 1 ml/min for 10 min) to right atrial neurons via their local arterial blood supply and during electrical stimulation of the stellate ganglia of open-chest anesthetized dogs. In AI dogs, ANG II caused ISF NE to increase fivefold (P < 0.05) without a significant increase in coronary sinus (CS) NE. Electrical stimulation (5 ms, 4 Hz, 8-14 V, and 10 min) of the stellate ganglia caused a similar increase in ISF NE (P < 0.05), accompanied by a sevenfold increase in CS NE (P < 0.05). ISF EPI increased greater than sixfold during ANG II infusion (P < 0.05) and during stellate stimulation. However, during ANG II infusions, aorta plasma EPI levels increased fourfold in AI dogs, whereas in AC dogs, CS NE and EPI levels were unaffected during ANG II infusions. Nevertheless, baseline ISF NE and EPI did not differ and increased to a similar extent during ANG II infusions in AI versus AC dogs. Thus exogenously administered ANG II increases the amount of NE liberated into the ISF independent of the adrenal contribution, the amount matching that induced by electrical stimulation of all cardiac sympathetic efferent neurons. In contrast, NE spillover into the CS occurred only during electrical stimulation of stellate ganglia. NE release and uptake mechanisms within the myocardium are differently affected, depending on how the final common pathway of the sympathetic efferent nervous system is modified.

RARbeta involvement in enhancement of lung tumor cell immunogenicity revealed by array analysis.

The retinoid receptors (RARs and RXRs) are mediators of the multiple effects of retinoic acid. Of these, the retinoic acid receptor beta2 (RARbeta2) has frequently been shown to be the principal mediator of the growth and tumor suppressive effects of retinoic acid; this gene is inactivated in many epithelial tumors and their derived cell lines. We have searched for genes that are regulated by this isoform and are potentially involved in tumor suppression. Using the Atlas human cDNA array I, we identified 27 genes (not counting RARbeta itself) that are regulated, directly or indirectly, by RARbeta2 when it is transfected into Calu-1, a lung tumor-derived line that does not normally express RARbeta. Several of the affected genes code for proteins whose functions would augment the process of apoptosis and/or the host's immune response. The latter group included ICAM-1 and MHC class I heavy chain, whose protein products play particularly important roles in the mounting of an effective anti-tumor response. We then confirmed by flow cytometry that the observed increases in message levels were reflected in increased cell surface protein levels for ICAM-1 and MHC class I in RARbeta2 transfectants of two RARbeta-deficient lines, Calu-1 and the epidermoid lung cancer-derived line SK-MES. Finally, we showed that RARbeta2 transfection of Calu-1 cells enhanced the heterologous CTL response in both the induction and the effector phases by up to threefold. These results support the hypothesis that down-regulation of these genes (and possibly others) in RARbeta-deficient tumor cells contributes to immune system evasion, and suggest a novel therapeutic approach for this disease.

Reflex innervation of the bulbocavernosus muscle.

OBJECTIVE: To determine the somatic reflex innervation of the bulbocavernosus muscle (BCM) and to illustrate the role of the perineal nerve in BCM contraction, using electrophysiological techniques. Subjects and methods Ten healthy, potent men underwent genital electrophysiological testing. Stimuli were applied to the dorsal nerve of the penis at the base of the penis, glans and anterior urethra, and the perineal nerve branch to the BCM. The electromyographic (EMG) responses of the BCM were recorded and onset latencies measured at baseline, both after anaesthetising the perineal nerve and after recovery from the anaesthetic. RESULTS: A reflex BCM contraction was elicited from all stimulation sites. Anaesthetizing the perineal nerve resulted in alterations of the EMG response. After the anaesthetic effect wore off, the responses returned to baseline values. CONCLUSIONS: Three distinct somatic bulbocavernosus reflexes were detected which are components of normal ejaculation. BCM contraction can be elicited after stimulating the dorsal penile nerve at the glans and the anterior urethra; these are the flexor responses of the bulbocavernosus reflex. BCM contraction can also be induced on stimulating the perineal nerve, which is the pathway through which the stretch and the tendon organ reflexes are carried to mediate muscle tone. The motor innervation of the BCM for all reflexes is carried through a branch of the perineal nerve. The findings may be relevant to the evaluation of ejaculatory disorders.

RARbeta2 specificity in mediating RA inhibition of growth of lung cancer-derived cells.

Retinoic acid receptor beta is the retinoid receptor most frequently associated with the growth suppressive effects of retinoic acid in various epithelial tumor-derived cell lines. In particular, it has been shown that transfection of RARbeta2 in epidermoid lung tumor cells could reduce their in vitro growth rate in the presence of retinoic acid and in vivo tumorigenicity. However, the question remained as to the isoform specificity of this effect. To investigate this, we transfected RARalpha1, RARbeta1 and RARbeta2 into the epidermoid lung cancer cell line Calu-1 and assessed the in vitro growth capacities of the transfected cells. The expression of the fetal RARbeta1 or overexpression of the ubiquitous RARalpha1 isoforms could not mimick the growth suppressive effect of RARbeta2. In addition we analyzed the expression of another RAR isoform, alpha2, in many tumor-derived lines and conclude from its expression pattern that RARalpha2 is unlikely to be involved in retinoic acid growth suppression of lung cancer. Overall our data suggest that the suppressive effect of RARbeta2 is isoform specific.

Cortical evoked responses from the perineal nerve.

PURPOSE: We characterize and determine normal reference latencies for the cortical evoked response from the perineal nerve. MATERIALS AND METHODS: A total of 15 healthy, potent men with a mean age of 31.3 +/- 6.8 years underwent genital electrophysiological testing. Square wave stimuli were applied to the perineal nerve at the ventral base of the penis and the perineal floor. Cortical evoked responses were recorded, and onset latencies were measured at baseline and after anesthetization of the dorsal nerve of the penis. RESULTS: In all 15 subjects a cortical evoked response was elicited after stimulation of the perineal nerve at the ventral base of the penis with a mean latency measured from the first positive deflection (P1) of 48.4 +/- 7.8 milliseconds. Penile block of the dorsal nerve of the penis did not change or abolish the cortical evoked response. A similar cortical evoked response was obtained, although less consistently, after stimulation of the perineal nerve through its course in the perineal floor. CONCLUSIONS: A cortical evoked response from the perineal nerve can be elicited reliably at the ventral penis. This neural pathway is independent of the dorsal nerve of the penis. The study of perineal somatic innervation may prove important in understanding the physiology of ejaculatory and erectile dysfunction.

Placebo-controlled trial of 13-cis-retinoic acid activity on retinoic acid receptor-beta expression in a population at high risk: implications for chemoprevention of lung cancer.

PURPOSE: To establish the incidence of abnormalities in the expression of retinoic acid receptor-beta (RARbeta) in bronchial cells and determine the capacity of 13-cis-retinoic acid (13-CRA) to correct such abnormalities. PATIENTS AND METHODS: One hundred eighty-eight smokers had a medical indication for bronchoscopy and were studied with bronchial brushings. Bronchial brushing samples were obtained for cytology analysis and for molecular analysis. After RNA was extracted, RARbeta sequences were amplified by reverse transcriptase polymerase chain reaction and Southern blots were performed to assess RARbeta expression. Forty-four eligible individuals with diminished RARbeta expression consented to double-blind randomization to receive a placebo or 13-CRA 30 mg orally daily for 6 months. A second bronchoscopy was performed at the end of the treatment period. An analysis of variance was used to analyze changes in RARbeta expression before and after treatment. RESULTS: The 6-month treatment course was completed by 27 patients, and results were obtained for a total of 18 patients (eight patients treated with 13-CRA and ten treated with the placebo). In the placebo group, there was no difference between the results of RARbeta expression before and after treatment (P =.43). In the 13-CRA group, there was an upregulation of RARbeta expression at the end of 13-CRA treatment (P =.001). Cytologic changes were uncommon. Toxicities were primarily of grade 1. Palatal brushings were compared with bronchial brushings in 40 smokers. A perfect correlation of the results of RARbeta expression was obtained from 27 patients. CONCLUSION: RARbeta expression is frequently decreased in the bronchial epithelium of smokers and is upregulated at the end of 13-CRA treatment. These results support undertaking a phase III chemoprevention trial of 13-CRA treatment for lung cancer.

Somatic innervation of the human bulbocavernosus muscle.

OBJECTIVE: To demonstrate the somatic reflex innervation of the bulbocavernosus muscle (BCM), the principal muscle for ejaculation. METHODS: Genitourinary electrodiagnostic testing utilizing modifications of the standard bulbocavernosus reflex was performed in 13 healthy male volunteers ages 20-43. RESULTS: Bulbocavernosus muscle contraction was elicited by stimulation of the dorsal nerve of the penis, from both the penile skin and from the anterior urethra, and following stimulation of the perineal nerve. Latencies were variable depending on the point of stimulation. CONCLUSIONS: All 3 afferent pathways synapse on pudendal motoneurons in the conus medullaris, and provide for peripheral reflex control of BCM contractions. Based on the latencies of the urethral evoked responses, urethral innervation differs from penile shaft innervation, each having a distinct population of the dorsal nerve of the penis (DNP) fibers. The presence of an electrically-defined pathway from the anterior urethra to the BCM suggests that somatic afferents from the anterior urethra are involved with the ejaculatory reflex. These somatic reflexes are components of normal ejaculatory function. The findings contribute to understanding the neurophysiology of ejaculation, and may be applicable to the evaluation of ejaculatory disorders.

Evidence for angiotensin-converting enzyme- and chymase-mediated angiotensin II formation in the interstitial fluid space of the dog heart in vivo.

BACKGROUND: We have previously demonstrated that angiotensin II (Ang II) levels in the interstitial fluid (ISF) space of the heart are higher than in the blood plasma and do not change after systemic infusion of Ang I. In this study, we assess the enzymatic mechanisms (chymase versus ACE) by which Ang II is generated in the ISF space of the dog heart in vivo. METHODS AND RESULTS: Cardiac microdialysis probes were implanted in the left ventricular (LV) myocardium (3 to 4 probes per dog) of 12 anesthetized open-chest normal dogs. ISF Ang I and II levels were measured at baseline and during ISF infusion of Ang I (15 micromol/L, n=12), Ang I+the ACE inhibitor captopril (cap) (2.5 mmol/L, n=4), Ang I+the chymase inhibitor chymostatin (chy) (1 mmol/L, n=4), and Ang I+cap+chy (n=4). ISF infusion of Ang I increased ISF Ang II levels 100-fold (P<0.01), whereas aortic and coronary sinus plasma Ang I and II levels were unaffected and were 100-fold lower than ISF levels. Compared with ISF infusion of Ang I alone, Ang I+cap (n=4) produced a greater reduction in ISF Ang II levels than did Ang I+chy (n=4) (71% versus 43%, P<0.01), whereas Ang I+cap+chy produced a 100% decrease in ISF Ang II levels. CONCLUSIONS: This study demonstrates for the first time a very high capacity for conversion of Ang I to Ang II mediated by both ACE and chymase in the ISF space of the dog heart in vivo.

Innervation of the human glans penis.

PURPOSE: We demonstrate the innervation of the glans penis through nerve blockade and electrophysiological tests. MATERIALS AND METHODS: The study was conducted in 14 healthy, sexually potent volunteers. The dorsal nerves of the penis were anesthetized bilaterally with lidocaine. Electrophysiological testing was performed by stimulating the dorsal nerve of the penis at the penile base distal to the block and recording action potentials at the glans. RESULTS: Dorsal nerve of the penis block resulted in anesthesia of the dorsal, lateral and glanular aspects of the penis. The ventral surface, including the frenulum, was intact to pinprick sensation. Dorsal nerve of the penis stimulation resulted in responses from the corona, dorsal and ventral mid glans, and penile shaft. Frenular responses were less consistently obtained. The most common recorded pattern was a monophasic waveform representing the arrival of a standing potential at a nerve terminal. Latencies were progressively longer with increasing distance from the point of stimulation with the longest latencies measured at the frenulum. Amplitudes of the responses decreased with increasing distance from the point of stimulation. CONCLUSIONS: The dorsal nerve of the penis innervates the glans, including the frenulum which is also innervated by a branch of the perineal nerve. Branches of the dorsal nerve of the penis extend through the glans ventrolaterally. Electrical representation of glanular innervation reveals the glans to be filled with nerve endings supporting its function as a sensory structure.

Mapping of chromosome 3p deletions in human epithelial ovarian tumors.

Epithelial ovarian tumors frequently display deletions on the short arm of chromosome 3 suggesting the existence of tumor suppressor genes within the deleted regions. We have recently established a primary tissue culture system as a model to investigate the genetic events associated with ovarian cancer. The frequencies of loss of heterozygosity (LOH) at 16 loci representative of chromosome 3p in 33 tumor biopsies and 47 ovarian primary cultures derived from unselected ovarian cancers were examined. This repertoire also included benign and borderline tumors as well as malignant ovarian ascites. LOH was observed in 25 (31%) samples for at least one marker: 21 of 58 malignant, two of 12 borderline and two of 10 benign specimens. Chromosome 3p loss was not restricted to ovarian tumors of high grade and stage. LOH was observed in both cultured and non cultured tumors and ascites. A spontaneously immortalized cell line derived from a malignant ovarian ascites, OV-90, displayed LOH of the majority of markers suggesting loss of one homolog of chromosome 3p. The pattern of deletion displayed by these 25 samples enabled the determination of at least two distinct regions of overlapping deletions on chromosome 3p extending from D3S1270 to D3S1597 and from D3S1293 to D3S1283. In addition, a region proximal to D3S1300 was deleted in a subset of samples. Although loss of loci overlapping these three regions (Regions I, II and III) were observed in malignant and benign tumors, in borderline tumors loss was observed of markers representative of Region III only. While RARbeta is presently included in Region II, the minimal regions of deletion exclude VHL, TGFBR2, PTPase(gamma) and FHIT as candidate tumor suppressors in ovarian tumorigenesis.

Human cerebrocortical potentials evoked by stimulation of the dorsal nerve of the penis.

Cortical evoked potentials resulting from stimulation of the dorsal nerve of the penis (DNP) provide a unique opportunity to document the cortical localization of sexual sensory representation in man. The DNP supplies sensory axons to the major portion of the human phallus, including the penile shaft and glans. Animal and human studies indicate that this nerve plays a crucial role in erection and ejaculation. Direct cortical evoked responses to DNP electrical stimulation were recorded in patients undergoing preoperative evaluation for resection of epileptic foci. These studies provided evidence that the primary sensory cortex contains a large area of cortex devoted to the afferent fibers of the DNP and that the sensory field is in a different location than previously described. The location and distribution of this response indicated the need for revision of the traditional concept of the sensory cortical homunculus.

Neuroanatomy of the penile portion of the human dorsal nerve of the penis.

OBJECTIVE: To determine the distribution of the dorsal nerve of the penis (DNP), the principal somatosensory nerve innervating the phallus, along the penile shaft and within the glans penis. MATERIALS AND METHODS: The DNP was dissected in human autopsy specimens, using low magnification, following the course of the nerve from the symphysis pubis into the glans penis. RESULTS: The shaft and glans were innervated by separate populations of axons. The lateral and ventral portions of the penile shaft were innervated by branches arcading from the dorsal midline radiating toward the ventral surface. These branches were very variable and some were seen to penetrate the corpus spongiosum. The axons innervating the glans were constant in a dorsal, midline location along most of the penile shaft, and once within the glans, there was abundant branching. Undulations in the nerves were noted along the penile shaft. CONCLUSIONS: The lateral arcading branches of the DNP provide a sensory pathway on the ventral and lateral penile shaft, and the termination of the fibres at the corpus spongiosum is consistent with pudendal innervation of the penile urethra. The distinct pattern of innervation of the glans emphasizes the role of the glans as a sensory structure. These findings may aid in planning penile surgical procedures.

Peripheral distribution of the human dorsal nerve of the penis.

PURPOSE: The integrity of the dorsal nerve of the penis is crucial for normal erectile and ejaculatory function. To our knowledge a description of this nerve along the phallus has not been formally described. We illustrate the distribution of the dorsal nerve of the penis to the penile shaft, anterior urethra and glans. MATERIALS AND METHODS: Neuroanatomical dissections were performed on 28 cadaver penis specimens. Electrodiagnostic testing was conducted on 10 healthy male subjects to confirm the anatomical findings. RESULTS: The dorsal nerve of the penis consists of 2 populations of axons, one to innervate the penile shaft and urethra, and the other to innervate the glans. Stimulation of the urethra resulted in responses recorded in the main trunk of the dorsal nerve of the penis and stimulation of the nerve evoked responses within the urethra. Bulbocavernosus muscle contraction was elicited following urethral stimulation. CONCLUSIONS: Urethral innervation by the dorsal nerve of the penis supports the view that urethral afferent impulses are a component of reflex ejaculatory activity. The pattern of glanular innervation by the dorsal nerve of the penis identifies the glans as a sensory end organ for sexual reflexes. The undulating character of the dorsal nerve of the penis is a mechanism by which the nerve can accommodate to significant changes in penile length with erection. Electrodiagnostic studies of the dorsal nerve of the penis should be modified to consider the anatomical findings.

Innervation of the human anterior urethra by the dorsal nerve of the penis.

Previous neuroanatomic studies have demonstrated that branches of the dorsal nerve of the penis (DNP) innervate the anterior urethra. This study utilized electrodiagnostic techniques to confirm this finding. Electrical stimulation of the urethra resulted in responses recorded in the main trunk of the DNP, and responses were recorded from the urethra following stimulation of the DNP. A bulbocavernosus reflex was evoked after urethral stimulation. Urethral afferent impulses have a role in reflex ejaculatory function.

The effect of pharmacologic erection on the dorsal nerve of the penis.

The dorsal nerve of the penis (DNP) is the primary source of afferent somatic input from the penis and is critical in the male sexual functions of erection and ejaculation. Using genitourinary electrodiagnostic techniques, this study was conducted to investigate the effect of pharmacologic erection on the DNP. Three tests were administered, and the changes in the DNP between flaccid, stretched, and erect states were examined. Calculated nerve conduction velocity (cNCV) measurements of the DNP increased with pharmacologic erection because mechanical straightening of the nerve allowed for a more precise measurement of nerve length. The latencies of the cortical evoked response and the bulbocavernosus reflex were not significantly changed with stretch or pharmacologic erection. In the evaluation of impotence, cNCV DNP measurements should be performed on the erect penis.