The relationship between rheumatoid arthritis and reproductive function

There is growing support for the theory that an interaction between the immune and reproductive/endocrine systems underlies the pathogenesis of autoimmune rheumatic diseases. Most of the recent evidence derives from studies of sex hormones and pregnancy in women with systemic lupus. Other than an ameliorative effect of pregnancy, little is known about reproductive factors in relation to rheumatoid arthritis. To elucidate the relationship, a population-based retrospective study was undertaken. Included were 378 female residents of Olmsted County, Minnesota diagnosed with rheumatoid arthritis between 1950 and 1982 (cases) and 325 arthritis-free, married female controls matched to the 324 married cases on birth-year, age at first marriage, and duration of Olmsted County residency. Information of reproductive factors was extracted from the medical records system maintained by the Mayo Clinic.^ Cases had lower fertility rates compared with the female population of Minnesota (rate ratio = 0.86, 95% confidence interval (CI)= 0.80-0.92). Fertility was significantly reduced even prior to the onset of rheumatoid factor positive arthritis. Restricting the comparison to married Olmsted County residents did not alter the results. Further adjustments for time not at risk of conception using survival analysis and proportional hazards modeling only intensified the fertility reduction in the married cases compared with controls. Nulligravidity was more common among cases than controls (odds ratio = 3.16, CI = 1.61-6.20). Independent of fertility, pregnancy had a protective effect against rheumatoid arthritis (odds ratio = 0.31, CI = 0.11-0.89), which was dramatically reversed in the 12 months postpartum (odds ratio = 4.67, CI = 1.50-14.47). Cases were younger at menopause than controls (p $<$ 0.01).^ Small but statistically insignificant associations were observed between rheumatoid arthritis and the following factors: increased frequency of complaints to a physician of infertility; increased frequency of spontaneous abortion, premature birth, and congenital malformations following arthritis onset; and increased prevalence of menopause at arthritis onset. Cases did not differ from controls on age at menarche, duration of pregnancy, or birth weight.^ The findings provide further support for the involvement of the reproductive/endocrine systems in the pathogenesis of autoimmune rheumatic disease. The search for biological mechanisms should be intensified. ^

Life and death of the embryonic female reproductive tract

Regardless of genetic sex, amniotes develop two sets of genital ducts, the Wolffian and Müllerian ducts. Normal sexual development requires the differentiation of one duct and the regression of the other. I show that cells in the rostral most region of the coelomic epithelium (CE) are specified to a Müllerian duct fate beginning at Tail Somite Stage 19 (TS19). The Müllerian duct (MD) invaginates from the CE where it extends caudally to and reaches the Wolffian duct (WD) by TS22. Upon contact, the MD elongates to the urogenital sinus separating the WD from the CE and its formation is complete by TS34. During its elongation, the MD is associated with and dependent upon the WD and I have identified the mechanism for MD elongation. Using the Rosa26 reporter to fate map the WD, I show that the WD does not contribute cells to the MD. Using an in vitro recombinant explant culture assay I show that the entire length of the MD is derived from the CE. Furthermore, I analyzed cell proliferation and developed an in vitro assay to show that a small population of cells at the caudal tip proliferates, laying the foundation for the formation of the MD. I also show that during its formation, the MD has a distinctive mesoepithelial character. The MD in males regresses under the influence of Anti-Müllerian Hormone (AMH). Through tissue-specific gene inactivation I have identified that Acvr1 and Bmpr1a and Smad1, Smad5 and Smad8 function redundantly in transducing the AMH signal. In females the MD differentiates into an epithelial tube and eventually the female reproductive tract. However, the exact tissue into which the MD differentiates has not been determined. I therefore generated a MD specific Cre allele that will allow for the fate mapping of the MD in both females males. The MD utilizes a unique form of tubulogenesis during development and to my knowledge is the only tubule that relies upon a signal from and the presence of another distinct epithelial tube for its formation.^

Dicer is required for female reproductive tract development and fertility in the mouse.

Dicer encodes a riboendonuclease required for microRNA biosynthesis. Dicer was inactivated in Müllerian duct mesenchyme-derived tissues of the reproductive tract of the mouse, using an Amhr2-Cre allele. Although Amhr2-Cre; Dicer conditional mutant males appeared normal and were fertile, mutant females were infertile. In adult mutant females, there was a reduction in the size of the oviducts and uterine horns. The oviducts were less coiled compared to controls and cysts formed at the isthmus near the uterotubal junction. Unfertilized, degenerate oocytes were commonly found within these cysts, indicating a defect in embryo transit. Beads transferred into the mutant oviduct failed to migrate into the uterus. In addition, blastocysts transferred directly into the mutant uterus did not result in pregnancy. Histological analysis demonstrated that the mutant uterus contained less glandular tissue and often the few glands that remained were found within the myometrium, an abnormal condition known as adenomyosis. In adult mutants, there was ectopic expression of Wnt4 and Wnt5a in the luminal epithelium (LE) and glandular epithelium (GE) of the uterus, and Wnt11 was ectopically expressed in GE. These results demonstrate that Dicer is necessary for postnatal differentiation of Müllerian duct mesenchyme-derived tissues of the female reproductive tract, suggesting that microRNAs are important regulators of female reproductive tract development and fertility.

Function and regulation of the Pem homeobox gene in vivo

Pem, a member of the PEPP homeobox family, is expressed in somatic cells in male and female reproductive tissues. In the adult murine testis, Pem is specifically expressed in Sertoli cells, where it is restricted to stages IV–VIII of the seminiferous epithelial cycle. To identify Pem's function in Sertoli cells, transgenic mice were generated that express Pem in Sertoli cells during all stages of the seminiferous epithelial cycle. This resulted in an increase in double-strand DNA breaks in preleptotene spermatocytes and single-strand DNA breaks in elongating spermatids. My results suggest that Pem regulates Sertoli-cell genes that encode secreted or cell-surface proteins that serve to control premeiotic DNA replication, DNA repair, and/or chromatin remodeling in the adjacent germ cells. Three additional transgenic mouse containing varying lengths of the Pem male-specific promoter (Pp) were generated to identify the sequences responsible for regulating Pem expression in the testis and epididymis. My analysis suggests that there are at least two regulatory regions in the Pem Pp. In the testis, region II directs androgen-dependent expression specifically in Sertoli cells whereas region I fine-tunes stage-specific expression by acting as a negative regulator. In the epididymis, region II confers androgen-dependent, developmentally-regulated expression in the caput whereas region I prevents inappropriate expression in the corpus. I also report the identification and characterization of two human PEPP family members related to Pem that I have named hPEPP1 and hPEPP2. The hPEPP1 and hPEPP2 homeodomains are more closely related to PEPP subfamily homeodomains than to any other homeodomain subfamily. Both genes are localized to the specific region of the human X chromosome that shares synteny with the region on the murine X chromosome containing three PEPP homeobox genes, Pem, Psx-1, and Psx-2. hPEPP1 and hPEPP2 mRNA expression is restricted to the testis but is aberrantly expressed in tumor cells of different origins, analogous to the expression pattern of Pem but not of Psx-1 or Psx-2. Unlike all known PEPP members, neither hPEPP1 nor hPEPP2 are expressed in placenta, which suggests that the regulation of the PEPP family has undergone significant alteration since the split between hominids and rodents. ^

Regulation of survivin gene expression in the human endometrium and endometrial cancer

In the United States, endometrial cancer is the leading cancer of the female reproductive tract. There are 40,100 new cases and 7,470 deaths from endometrial cancer estimated for 2008 (47). The average five year survival rate for endometrial cancer is 84% however, this figure is substantially lower in patients diagnosed with late stage, advanced disease and much higher for patients diagnosed in early stage disease (47). Endometrial cancer (EC) has been associated with several risk factors including obesity, diabetes, hypertension, previously documented occurrence of hereditary non-polyposis colorectal cancer (HNPCC), and heightened exposure to estrogen (25). As of yet, there has not been a dependable molecular predictor of endometrial cancer occurrence in women with these predisposing factors. The goal of our lab is to identify genes that are aberrantly expressed in EC and may serve as molecular biomarkers of EC progression. One candidate protein that we are exploring as a biomarker of EC progression is the cell survival protein survivin.

Prevalence of Premature Ovarian Failure in Women with Tuberous Sclerosis

Tuberous Sclerosis Complex (TSC) is an autosomal dominant tumor suppressor disorder characterized by hamartomas, or benign growths, in various organ systems. Inactivating mutations in either the TSC1 or the TSC2 gene cause most cases of TSC. Recently, the use of ovarian specific conditional knock-out mouse models has demonstrated a crucial role of the TSC genes in ovarian function. Mice with complete deletion of Tsc1 or Tsc2 showed accelerated ovarian follicle activation and subsequent premature follicular depletion, consistent with the human condition premature ovarian failure (POF). POF is defined in women as the cessation of menses before the age of 40 and elevated levels of follicle stimulating hormone (FSH). The prevalence of POF is estimated to be 1%, affecting a substantial number of women in the general population. Nonetheless, the etiology of most cases of POF remains unknown. Based on the mouse model results, we hypothesized that the human TSC1 and TSC2 genes are likely to be crucial for ovarian development and function. Moreover, since women with TSC already have one inactivated TSC gene, we further hypothesized that they may show a higher prevalence of POF. To test this hypothesis, we surveyed 1000 women with TSC belonging to the Tuberous Sclerosis Alliance, a national support organization. 182 questionnaires were analyzed for information on menstrual and reproductive function, as well as TSC. This self-reported data revealed 8 women (4.4%) with possible POF, as determined by menstrual history report and additional supportive data. This prevalence is much higher than 1% in the general population. Data from all women suggested other reproductive pathology associated with TSC such as a high rate of miscarriage (41.2%) and menstrual irregularity of any kind (31.2%). These results establish a previously unappreciated effect of TSC on women’s reproductive health. Moreover, these data suggest that perturbations in the cellular pathways regulated by the TSC genes may play an important role in reproductive function.

Modulatory effects of bag cell peptides in {\it Aplysia}: Behavioral and electrophysiological studies

Reproductive hormones have effects on the nervous system not directly related to reproductive function. In the rat, for example, luteinizing hormone releasing hormone has dramatic effects on learning and memory. The present work attempts to examine the effects of reproductive hormones on non-reproductive behaviors and the neural loci and mechanisms underlying these effects in Aplysia, an animal whose behaviors, reproductive hormones and neural circuitry have been well characterized.^ In Aplysia, the neurosecretory bag cells release several peptides that are responsible for eliciting egg laying. The effects of these peptides on the defensive tail-siphon withdrawal reflex, as well as sensitization of this reflex, were examined. Sensitization, a simple form of nonassociative learning, refers to the behavioral enhancement of a response to a test stimulus after the presentation of a strong stimulus, that may last minutes (short-term) or days (long-term). An extract of the bag cells (BCE) inhibited the baseline siphon component of the tail-siphon withdrawal reflex and suppressed long-term, but not short-term, sensitization of the reflex. Preliminary experiments suggest that BCE also affects the tail component of the tail-siphon withdrawal reflex.^ To determine the neural mechanisms underlying the inhibition of the baseline reflex, electrophysiological studies were performed using an in vitro analogue of the tail-siphon withdrawal reflex to examine the ability of BCE, as well as the individual bag cell peptides (BCPs), to modulate the circuitry of the reflex. Bag cell extract attenuated the synaptic strength of the monosynaptic connections between tail sensory neurons and tail motor neurons. When individually applied only $\beta$-BCP produced a similar attenuation. This effect of $\beta$-BCP was not dependent on changes in duration of the presynaptic action potential.^ An in vitro analogue of long-term sensitization training was developed to examine the mechanisms by which the BCPs may affect long-term sensitization of the tail-siphon withdrawal reflex. This analogue exhibited both short- and long-term facilitation of the connections between the tail sensory and motor neurons.^ The results of these behavioral and electrophysiological experiments suggest that the BCPs inhibit the tail-siphon withdrawal reflex, at least in part, by modulating the synaptic strength of the connections between the sensory neurons and motor neurons underlying the reflex. One candidate for this effect is $\beta$-BCP. Thus, the peptides which elicit egg laying may also serve other functions such as the inhibition of defensive reflexes. In addition, these experiments raise the possibility that BCPs may exert a long lasting effect ($>$24 hr), suppressing long-term sensitization of the tail-siphon withdrawal reflex. ^

Investigation of the molecular mechanisms of endometrial hyperplasia

While there is considerable information on the molecular aberrations associated with the development of endometrial cancer, very little is known of changes in gene expression associated with its antecedent premalignant condition, endometrial hyperplasia. In order to address this, we have compared the level of expression of components of the IGF-I signaling pathway in human endometrial hyperplasia to their level of expression in both the normal pre-menopausal endometrium and endometrial carcinoma. We have also characterized the molecular characteristics of endometrial hyperplasia as it occurs in a murine model of hormone-dependent tumorigenesis of the female reproductive tract. ^ There was a significant and selective increase in the expression of the IGF-I Receptor (IGF-IR) in both human hyperplasia and carcinoma as compared to the normal endometrium. The receptor was also activated, as judged by increased tyrosine phosphorylation. In addition, in hyperplasia and carcinoma there is activation of the downstream component Akt. The expression of the PTEN tumor suppressor is decreased in a subset of subjects with hyperplasia and in all of the carcinomas. The simultaneous loss of PTEN expression and increased IGF-IR activation in the hyperplastic endometrium was associated with an increased incidence of endometrial carcinoma elsewhere within the uterus. In the rodent hyperplasia, there was a significant increase in the expression and activation of Akt that appears to be attributable to a marked increase in the expression of IGF-II. ^ Our studies have demonstrated the pathologic proliferation of both the human and rodent endometrium is linked to a marked activation of the Akt pathway. However the cause of this dysregulation is different in the human disease and the animal model. In rodents, hyperplasia is linked to increased expression of one of the ligands of the IGF-IR, IGF-II. In humans the IGF-I receptor itself is upregulated and activated. Additional activation of the Akt pathway via the suppression of PTEN activity, results in conditions that are associated with the marked increase in the probability of developing endometrial cancer. Our data suggests that increased activity of the IGF-I pathway plays the key role in the hyperproliferative state characteristic of endometrial hyperplasia and cancer.^

Estrogen-induced gene expression and patient survival in high-grade serous carcinoma of the ovary

Background. Assessment of estrogen receptor (ER) expression has inconsistent utility as a prognostic marker in epithelial ovarian carcinoma. In breast and endometrial cancers, the use of estrogen-induced gene panels, rather than ER expression alone, has shown improved prognostic capability. Specifically, over-expression of estrogen-induced genes in these tumors is associated with a better prognosis and signifies estrogen sensitivity that can be exploited with hormone antagonizing agents. It was therefore hypothesized that estrogen-induced gene expression in ovarian carcinoma would successfully predict outcomes and differentiate between tumors of varying estrogen sensitivities. Methods. Two hundred nineteen (219) patients with ovarian cancer who underwent surgery at M. D. Anderson between 2004 and 2007 were identified. Of these, eighty-three (83) patients were selected for inclusion because they had advanced stage, high-grade serous carcinoma of the ovary or peritoneum, had not received neoadjuvant chemotherapy, and had readily available frozen tissue for study. All patients had also received adjuvant treatment with platinum and taxane agents. The expression of seven genes known to be induced by estrogen in the female reproductive tract (EIG121, sFRP1, sFRP4, RALDH2, PR, IGF-1, and ER) was measured using qRT-PCR. Unsupervised cluster analyses of multiple gene permutations were used to categorize patients as high or low estrogen-induced gene expressors. QPCR gene expression results were then compared to ER and PR immunohistochemical (IHC) expression. Cox proportional hazards models were used to evaluate the effects of both individual genes and selected gene clusters on patient survival. Results. Median follow-up time was 38.7 months (range 1-68 months). In a multivariate model, overall survival was predicted by sFRP1 expression (HR 1.10 [1.02-1.19], p=0.01) and EIG121 expression (HR 1.28 [1.10-1.49], p<0.01). A cluster defined by EIG121 and ER was further examined because that combination appeared to reasonably segregate tumors into distinct groups of high and low estrogen-induced gene expressors. Shorter overall survival was associated with high estrogen-induced gene expressors (HR 2.84 [1.11-7.30], p=0.03), even after adjustment for race, age, body mass index, and residual disease at debulking. No difference in IHC ER or PR expression was noted between gene clusters. Conclusion. In sharp contrast to breast and endometrial cancers, high estrogen-induced gene expression predicts shorter overall survival in patients with high-grade serous ovarian carcinoma. An estrogen-induced gene biomarker panel may have utility as prognostic indicator and may be useful to guide management with estrogen antagonists in this population.^

ROLE OF SOX9 IN UTERINE GLAND DEVELOPMENT AND DISEASE INITIATION

The female reproductive tract (FRT) develops midway through embryogenesis, and consists of oviducts, uterine horns, cervix and upper part of the vagina. The uterine horns are composed of an epithelial layer, luminal (LE) and glandular epithelium (GE), surrounded by a mesenchymal layer, the stroma and myometrium. Interestingly, in most mammals the GE forms after birth and it only becomes fully differentiated as the female reaches sexual maturity. Uterine glands (UG) are made up of GE and are present in all mammals. They secrete nutrients, cytokines and several other proteins, termed histotroph, that are necessary for embryo implantation and development. Experiments in ewes and mice have revealed that females who lack UGs are infertile mainly due to impaired implantation and early pregnancy loss, suggesting that UGs are essential for fertility. Fortunately for us, UGs develop after birth allowing us to peer into the genetic mechanism of tubulogenesis and branching morphogenesis; two processes that are disrupted in various adenocarcinomas (cancer derived from glands). We created 3D replicas of the epithelium lining the FRT using optical projection tomography and characterized UG development in mice using lineagetracing experiments. Our findings indicate that mouse UGs develop as simple tubular structures and later grow multiple secretory units that stem from the main duct. The main aim of this project was to study the role of SOX9 in the UGs. Preliminary studies revealed that Sox9 is mostly found in the nucleus of the GE. vii This observation led to the hypothesis that Sox9 plays a role in the formation and/or differentiation of the GE. To study the role of Sox9 in UGs differentiation, we conditionally knocked out and overexpressed Sox9 in both the LE and GE using the progesterone receptor (Pgr) promoter. Overexpressing Sox9 in the uterine epithelium, parts of the stroma, and myometrium led to formation of multiple cystic structures inside the endometrium. Histological analysis revealed that these structures appeared morphologically similar to structures present in histological tissue sections obtained from patients with endometrial polyps. We have accounted for the presence of simple and complex hyperplasia with atypia, metaplasia, thick-walled blood vessels, and stromal fibrosis; all “hallmarks” that indicate overexpressing Sox9 leads to development of a polyp-like morphology. Therefore, we can propose the use of Sox9-cOE mice to study development of endometrial cystic lesions and disease progression into hyperplastic lesions.

GENETIC ANALYSIS OF THE FUNCTION OF THE DROSOPHILA DOUBLESEX-RELATED FACTOR dmrt93B

DMRT (Doublesex and Mab-3 related transcription factor) proteins generally associated with sexual differentiation in many organisms share a common DNA binding domain and are often expressed in reproductive tissues. Aside from doublesex, which is a central factor in the regulation of sex determination, Drosophila possesses three different dmrt genes that are of unknown function. Because the association with sexual differentiation and reproduction is not universal and some DMRT proteins have been found to play other developmental roles we chose to further characterize one of these Drosophila genes. We carried out genetic analysis of dmrt93B, which was previously found to be expressed sex-specifically in the developing somatic gonad and to affect testis morphogenesis in RNAi knockdowns. In order to disrupt this gene, the GAL4 yeast transcriptional activator followed by a polyadenylation signal was inserted after the dmrt93B start codon and introduced into the genome by homologous recombination. Analysis of the knock-in mutation as well as a small deletion removing all dmrt93B sequence demonstrate that loss of function causes partial lethality at the late pupal stage. Surprisingly, these mutations have no significant effect on gonad formation or male fertility. Analysis of GAL4-driven GFP reporter expression indicates that the dmrt93B promoter activity is highly specific to neurons in the suboesophageal and proventricular ganglion in larva and adult of both sexes suggesting a possible role in digestive tract function. Using the Capillary Feeder (CAFÉ) assay to measure daily food intake we find that reduction in this gene’s function leads to an increase in food consumption. These results suggest dmrt93 plays an important role in the formation or maintenance of neurons that affect feeding and support the idea that dmrt genes may not be restricted to roles in sexual differentiation.

Effects of hypoxic-ischemic encephalopathy and whole-body hypothermia on neonatal auditory function: a pilot study.

We assessed the effects of hypoxic-ischemic encephalopathy (HIE) and whole-body hypothermia therapy on auditory brain stem evoked responses (ABRs) and distortion product otoacoustic emissions (DPOAEs). We performed serial assessments of ABRs and DPOAEs in newborns with moderate or severe HIE, randomized to hypothermia ( N = 4) or usual care ( N = 5). Participants were five boys and four girls with mean gestational age (standard deviation) of 38.9 (1.8) weeks. During the first week of life, peripheral auditory function, as measured by the DPOAEs, was disrupted in all nine subjects. ABRs were delayed but central transmission was intact, suggesting a peripheral rather than a central neural insult. By 3 weeks of age, peripheral auditory function normalized. Hypothermia temporarily prolonged the ABR, more so for waves generated higher in the brain stem but the effects reversed quickly on rewarming. Neonatal audiometric testing is feasible, noninvasive, and capable of enhancing our understanding of the effects of HIE and hypothermia on auditory function.

Postoperative renal function preservation with nonischemic femoral arterial cannulation for thoracoabdominal aortic repair.

BACKGROUND: Renal failure after thoracoabdominal aortic repair is a significant clinical problem. Distal aortic perfusion for organ and spinal cord protection requires cannulation of the left femoral artery. In 2006, we reported the finding that direct cannulation led to leg ischemia in some patients and was associated with increased renal failure. After this finding, we modified our perfusion technique to eliminate leg ischemia from cannulation. In this article, we present the effects of this change on postoperative renal function. METHODS: Between February 1991 and July 2008, we repaired 1464 thoracoabdominal aortic aneurysms. Distal aortic perfusion was used in 1088, and these were studied. Median patient age was 68 years, and 378 (35%) were women. In September 2006, we began to adopt a sidearm femoral cannulation technique that provides distal aortic perfusion while maintaining downstream flow to the leg. This was used in 167 patients (15%). We measured the joint effects of preoperative glomerular filtration rate (GFR) and cannulation technique on the highest postoperative creatinine level, postoperative renal failure, and death. Analysis was by multiple linear or logistic regression with interaction. RESULTS: The preoperative GFR was the strongest predictor of postoperative renal dysfunction and death. No significant main effects of sidearm cannulation were noted. For peak creatinine level and postoperative renal failure, however, strong interactions between preoperative GFR and sidearm cannulation were present, resulting in reductions of postoperative renal complications of 15% to 20% when GFR was <60 mL>/min/1.73 m(2). For normal GFR, the effect was negated or even reversed at very high levels of GFR. Mortality, although not significantly affected by sidearm cannulation, showed a similar trend to the renal outcomes. CONCLUSION: Use of sidearm cannulation is associated with a clinically important and highly statistically significant reduction in postoperative renal complications in patients with a low GFR. Reduced renal effect of skeletal muscle ischemia is the proposed mechanism. Effects among patients with good preoperative renal function are less clear. A randomized trial is needed.

A study of factors regulating the autonomous proliferation of the steroid hormone-induced LJ6195 murine reproductive tract tumor

Neonatal estrogen treatment of BALB/c mice results in the unregulated proliferation of the cervicovaginal epithelium and eventually tumorigenesis. The conversion of the normally estrogen responsive cyclic proliferation of the vaginal epithelium to a continuous estrogen-independent pattern of growth is a complex phenomenon. The aim of this study was to gain an understanding of the mechanism(s) by which steroid hormone administration during a critical period of development alters the cyclic proliferation of vaginal epithelium, ultimately leading to carcinogenesis in the adult animal.^ The LJ6195 murine cervicovaginal tumor was induced by treating newborn female BALB/c mice with 20 $\mu$g 17$\beta$-estradiol plus 100 $\mu$g progesterone for the first 5 days after birth. In contrast to proliferation of the normal vaginal epithelium, proliferation of LJ6195 is not regulated by estradiol. Northern blot analysis of RNA from vaginal tracts of normal mice, neonatal-estrogen treated mice, and LJ6195 indicate that there is an alteration in the expression of several genes such as the estrogen receptor, c-fos, and HER2/neu. In response to neonatal estrogen treatment, the estrogen receptor is down regulated in the murine vaginal tract. Therefore, the estrogen-independent nature of this tissue is established as early as 3 months after treatment. There is strong evidence that the proliferation of LJ6195 is regulated through an autocrine growth pathway. The LJ6195 tumor expresses mRNA for the epidermal growth factor receptor. In addition, conditioned medium from the LJ6195 tumor cell line contains a growth factor(s) with epidermal growth factor-like activity. Conditioned medium from the LJ6195 cell line stimulated the proliferation of the EGF-dependent COMMA D mouse mammary gland cell line in a dose-dependent manner. The addition of an anti-mEGF-antibody to LJ6195 cell cultures significantly decreased growth. These results suggest that the EGF-receptor mediated growth pathway may play a role in regulating the estrogen-independent proliferation of the LJ6195 tumor. ^

CHARACTERIZATION OF CYTOCHROME P-450 MIXED FUNCTION OXIDASE OF RAT COLON MUCOSA

Non-pregnant, female adult rats pretreated with either phenobarbital (PB) or (beta)-naphthoflavone ((beta)NF) through short-course intraperitoneal injections were shown by sodium dithionite-reduced carbon monoxide difference spectroscopy and NADPH-cytochrome c in vitro assay to contain cytochrome P-450 and NADPH-dependent reductase associated with the microsomal fraction of colon mucosa. These two protein components of the mixed function oxidase system were released from the microsomal membrane, resolved from each other, and partially purified by using a combination of techniques including solubilization in nonionic detergent followed by ultracentrifugation, anion exchange and adsorption column chromatographies, native gel electrophoresis, polyethylene glycol fractionation and ultrafiltration.^ In vitro reconstitution assays demonstrated the cytochrome P-450 fraction as the site of substrate and molecular oxygen binding. By the use of immunochemical techniques including radial immunodiffusion, Ouchterlony double diffusion and protein electroblotting, the cytochrome P-450 fraction was shown to contain at least 5 forms of the protein, having molecular weights as determined by SDS gel electrophoresis identical to the corresponding hepatic cytochrome P-450. Estimation of total cytochrome P-450 content confirmed the preferential induction of particular forms in response to the appropriate drug pretreatment.^ The colonic NADPH-dependent reductase was isolated from native gel electrophoresis and second dimensional SDS gel electrophoresis was performed in parallel to that for purified reductase from liver. Comparative electrophoretic mobilities together with immunochemical analysis, as with the cytochrome P-450s, reconstitution assays, and kinetic characterization using artificial electron acceptors, gave conclusive proof of the structural and functional homology between the colon and liver sources of the enzyme.^ Drug metabolism was performed in the reconstituted mixed function oxidase system containing a particular purified liver cytochrome P-450 form or partially pure colon cytochrome P-450 fraction plus colon or liver reductase and synthetic lipid vesicles. The two drugs, benzo{(alpha)}pyrene and benzphetamine, which are most representative of the action of system in liver, lung and kidney, were tested to determine the specificity of the reconstituted system. The kinetics of benzo{(alpha)}pyrene hydroxylation were followed fluorimetrically for 3-hydroxybenzo{(alpha)}pyrene production. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^