David S. Grosso

CHARACTERIZATION OF A NEW HUMAN ENDOMETRIAL CARCINOMA (RL95-2) ESTABLISHED IN TISSUE CULTURE

SummaryA new human endometrial cell line, RL95-2, derived from a Grade 2 moderately differentiated adenosquamous carcinoma of the endometrium has been passaged successfully in cell culture for more than 2 yr. The cells are characteristically epithelioid with well-defined junctional complexes, tonofilaments, filopodialike extensions, and surface microvilli. Nuclei are large, irregular, and invaginated frequently with multiple, prominent, lamellar nucleoli. The cells have a log phase doubling time of 22 to 34 h followed by continued growth at a reduced rate with no apparent plateau phase. They exhibit a strong tendency for piling up as well as for the formation of glandlike dome structures. Karyotypically the line is trisomic 8 (47,XX,+8) and has an 8% frequency of polyploidization. Both cytoplasmic and nuclear estrogen receptors are present. Antihuman α-keratin characterizes the cell line as epithelial, nonstromal. The RL95-2 cell line may provide a useful in vitro system for the investigation of the endocrine regulation of endometrial neoplasia.

Pineal melatonin inhibition of tumor promotion in the N-nitroso-N-methylurea model of mammary carcinogenesis : potential involvement of antiestrogenic mechanisms in vivo

SummaryTheN-methyl-N-nitrosurea (NMU) model of hormone-responsive rat mammary carcinogenesis was used to address the hypothesis that melatonin (Mel), the principle hormone of the pineal gland, inhibits tumorigenesis by acting as an anti-promoting rather than an anti-initiating agent. Daily late-afternoon injections of Mel (500 μg/day), restricted to the initiation phase of NMU mammary tumorigenesis, were ineffective in altering tumor growth over a 20-week period. When Mel treatment was delayed for 4 weeks after NMU and then continued through the remainder of the promotion phase, only tumor number was significantly lower than in controls. However, when Mel injections encompassed the entire promotion phase, both tumor incidence and number were significantly lower than in the controls. Although elimination of the endogenous Mel signal via pinealectomy promoted tumor growth, the effect was not statistically significant. Serum levels of estradiol and tumor estrogen receptor content were unaltered by either Mel or pinealectomy. While Mel treatment failed to affect circulating prolactin levels, pinealectomy caused a two-fold increase in serum prolactin. The estradiol-stimulated recrudescence of tumors following ovariectomy was completely blocked by either 20, 100 or 500 μg Mel/day or tamoxifen (20 μg/day). Thus, Mel appears to be an antipromoting hormone that may antagonize the tumor-promoting actions of estradiol in this model of mammary tumorigenesis.

Tamoxifen therapy in recurrent epithelial ovarian carcinoma

Thirty-seven patients with recurrent epithelial ovarian carcinoma were entered into a trial of tamoxifen therapy (10 mg BID) to determine the effect on long-term survival. Thirty-one patients were evaluable with follow-up ranging from 6 to 42 months since initiation of hormonal therapy. All patients were heavily pretreated with multiple chemotherapeutic regimens (median 3). There was 1 complete responder (3.2%), 2/31 (6.4%) had a partial response, 6/31 (19.3%) had stable disease, and 22/31 (71%) had progressive disease. Twenty-four patients are dead (23 from advanced carcinoma, 1 from cardiac causes); 5 patients are alive with disease; 2 patients are lost to follow-up. Median survival of nonresponders was 7 months versus 16 months for responders (CR + PR + stable disease) (P = 0.001 life table analysis). Of the 9 responders, 7 had poorly differentiated tumors (grades 3 or 4), and 2 had moderately differentiated tumors (grade 2). Eleven patients had estrogen and progesterone receptor studies (ER, PR). No correlation between response rate and receptor status was evident. We conclude that although significant disease regression is unlikely to result from tamoxifen therapy, there may be a subset of patients who can benefit from the cytostatic properties of hormonal manipulation.

Isolation and characterization of myocytes from the adult rat heart.

Spontaneously contracting myocytes were isolated from ventricles of the adult rat heart. Hearts were perfused retrogradally via the aorta for 30 minutes at 37 degrees C with Ca2+-free phosphate-buffered saline containing collagenase and hyaluronidase. The venticles were divided into pieces and incubated 15 minutes with the enzymes. Dislodged cells were decanted, diluted with cold buffer and allowed to settle. The washed cells were then sedimented through 3% Ficoll. This procedure yielded approximately 50 mg of protein from 1 gm of heart. Viability measured by trypan-glue exclusion is 90-95%. Approximately 80% of the cells were beating. Scanning electron microscopic studies suggest that the isolated myocytes are morphologically intact. The cells oxidize glucose, pyruvate, citrate and palmitate to CO2 and synthesize protein and RNA. Uptake of glucose, 2-deoxyglucose, leucine and taurine was saturable. Glucose uptake was stimulated by insulin. The cells retained LDH and CPK as well as their capacity to oxidize substrates after 24 hours at 4 degrees C or 4 hours at 37 degrees C. After 24 hours at 4 degrees C the cells resume contracting when returned to room temperature. The procedure reported here for the isolation of spontaneously contracting, adult, rat heart myocytes provides cells with a high index of viability and greater yield than previously reported methods. The cells retain metabolic activity and withstand storage for longer periods than other described preparations.

Ketoconazole Inhibition of Testicular Secretion of Testosterone and Displacement of Steroid Hormones from Serum Transport Proteins

In vivo perfusion of canine testes with ketoconazole inhibited the stimulation of testosterone production by human chorionic gonadotropin in a dose-dependent manner. Ketoconazole also selectively displaced steroids from serum-binding globulins. Dihydrotestosterone and estradiol binding to sex hormone-binding globulin were inhibited by ketoconazole. Cortisol binding to corticosteroid-binding globulin was unaffected. The concentrations of ketoconazole that inhibited human chorionic gonadotropin stimulation of testicular androgen production and displaced sex steroids from sex hormone-binding globulin were in the range of blood levels found in patients on higher therapeutic dosage regimens. Suppression of testicular testosterone synthesis and displacement of estrogens from sex hormone-binding globulin may decrease the androgen/estrogen ratio of the blood and contribute to the development of gynecomastia that has been reported in some ketoconazole-treated patients.

Ultrastructural studies of metabolically active isolated adult rat heart myocytes

Abstract Myocytes from adult rat heart were isolated by a method recently developed by us in which hearts were pre-perfused with calcium-free phosphate-buffered saline medium containing collagenase and hyaluronidase. This was followed by incubation in the enzyme medium and cellular sedimentation through 3% Ficoll. Myocytes isolated in this manner were metabolically active and morphologically intact. In the present study, scanning and transmission electron microscopy of isolated myocytes showed long cylindrical cells with transverse microridges which corresponded directly with sarcomere lengths. Most cells appeared to be in a fully contracted state. Contractile elements and associated membranes, other intracellular compartments and sarcolemmae were indistinguishable from their in vivo counterparts. Although myocytic basal laminae and other structurally identifiable surface coats were removed by our isolation procedure, sarcolemmae remained remarkably unaffected. Cyclic AMP assays in control and epinephrine- or glucagon-stimulated cells strongly suggested that membranebound receptors were present and the functional integrity of the sarcolemmae was maintained in our preparations.

The Maternal Immune Response in Coccidioidomycosis: Is Pregnancy a Risk Factor for Serious Infection?

Seven subjects with prior coccidioidal disease and three with active Coccidioides immitis infection during their first trimester were studied during pregnancy and postpartum to determine their general and antigen-specific cell-mediated immune status. All ten were white and carried their pregnancies to term without incident. Decreases in total lymphocytes and T-helper and T-suppressor subsets were noted during the third trimester, presumably secondary to an increase in plasma volume. Lymphocyte responses to the mitogens phytohemagglutinin, concanavalin A, and pokeweed were mildly decreased late in pregnancy, with significant intrasubject and intersubject variation. Responses to tetanus antigen were consistently and significantly lower as pregnancy progressed, rising above first trimester levels by 12 weeks postpartum. A similar pattern of response was noted with spherulin antigen for the seven subjects with previously demonstrated coccidioidal immunity. The three subjects with active coccidioidomycosis either failed to mount a significant spherulin immune response or demonstrated an early response that fell as pregnancy progressed. This antigen-specific immune suppression continued for up to 16 months postpartum despite the fact that there was no clinical evidence of coccidioidal activity beyond the first trimester. Thus, while all three completed pregnancy without complication, the data suggest that significantly increased maternal risk may be present when active coccidioidomycosis and pregnancy occur together. This risk may be greatest among darker-skinned individuals who become infected during the latter half of pregnancy.

Characterization of a carrier-mediated transport system for taurine in the fetal mouse heart in vitro.

Cardiac taurine levels are elevated in hypertension and congestive heart failure. A possible mechanism for this increase in taurine is an alteration of its uptake. We sought to identify and characterize a carrier-mediated transport system for taurine in the mammalian myocardium utilizing the fetal mouse heart in organ culture. Hearts from fetuses of 16-19 days gestational age used in these studies had an endogenous taurine content of 14.1+/-0.5 nmol/mg tissue. The uptake of [(3)H]taurine was linear for up to 8 h. Taurine was accumulated against a concentration gradient as demonstrated by a net increase in taurine concentration when hearts were incubated in 0.5 mM taurine. [(3)H]Taurine uptake was saturable, K(m) = 0.44 mM, temperature dependent, and required sodium. The close structural analogues, hypotaurine and beta-alanine, reduced [(3)H]taurine uptake by 87% when present in 100-fold excess. The alpha-amino acids alanine, alpha-aminoisobutyric acid, glycine, leucine, and threonine did not inhibit uptake. Other taurine analogues tested were guanidinotaurine, guanidinopropionic acid, gamma-aminobutyric acid, 2-aminoethane phosphonic acid, aminomethane sulfonic acid, 3-aminopropane sulfonic acid, N-acetyltaurine, and isethionic acid. We conclude that a carrier-mediated transport system for taurine exists in the fetal mouse heart based on the demonstration of (a) temperature dependence, (b) saturability, and (c) structural selectivity of the uptake process. Transport was demonstrated to be mediated by a beta-amino acid uptake system. In addition, taurine uptake was observed to be sodium dependent, energy dependent, and capable of accumulating taurine against a concentration gradient.

Melatonin Action on Oncogenesis

Great strides have been made within the last decade in our understanding of the processes involved in oncogenesis not only at the organismal level but at the cellular-molecular level as well. It has become clear that hormones and growth factors play a prominent role in the mechanisms governing oncogenesis. The importance of the hormonal and growth factor regulation of oncogenesis is particularly evident with respect to cancer of the breasts1.

Clinical InvestigationsThe Maternal Immune Response in Coccidioidomycosis: Is Pregnancy a Risk Factor for Serious Infection?

Seven subjects with prior coccidioidal disease and three with active Coccidioides immitis infection during their first trimester were studied during pregnancy and postpartum to determine their general and antigen-specific cell-mediated immune status. All ten were white and carried their pregnancies to term without incident. Decreases in total lymphocytes and T-helper and T-suppressor subsets were noted during the third trimester, presumably secondary to an increase in plasma volume. Lymphocyte responses to the mitogens phytohemagglutinin, concanavalin A, and pokeweed were mildly decreased late in pregnancy, with significant intrasubject and intersubject variation. Responses to tetanus antigen were consistently and significantly lower as pregnancy progressed, rising above first trimester levels by 12 weeks postpartum. A similar pattern of response was noted with spherulin antigen for the seven subjects with previously demonstrated coccidioidal immunity. The three subjects with active coccidioidomycosis either failed to mount a significant spherulin immune response or demonstrated an early response that fell as pregnancy progressed. This antigen-specific immune suppression continued for up to 16 months postpartum despite the fact that there was no clinical evidence of coccidioidal activity beyond the first trimester. Thus, while all three completed pregnancy without complication, the data suggest that significantly increased maternal risk may be present when active coccidioidomycosis and pregnancy occur together. This risk may be greatest among darker-skinned individuals who become infected during the latter half of pregnancy.