Alan L. Lin

Estradiol-17 beta affects estrogen receptor distribution and elevates progesterone receptor content in baboon aorta.

We used the synthetic estrogen R2858 (moxestrol) and estradiol-170, respectively, to characterize the estrogen receptor in baboon (Papio sp.) aortic or myocardial cytoplasmic and nuclear preparations. We observed regional differences in the cytoplasmic fraction estrogen and progesterone receptor content of aortic arch, thoracic aorta, and abdominal aorta when tissues from either oophorectomized or oophorectomized estradiol-170-treated subjects were compared. The estrogen receptor content was highest in the abdominal aorta and lowest in the aortic arch. In contrast, the cytoplasmic fraction progesterone receptor content was highest in the aortic arch and lowest in the abdominal aorta. The nuclear fraction estrogen receptor could not be demonstrated in preparations from cardiovasculature of oophorectomized female baboons. The use of Silastic implants to administer a physiologic concentration of estradiol-170 to oophorectomized female baboons caused a 20% to 50% reduction in cytoplasmic fraction estrogen receptor content, which was quantitatively accounted for by the appearance of estrogen receptor in the corresponding nuclear aortic or myocardial preparation. Estrogen administration caused a 20% to 40% increase inc cytoplasmic fraction progesterone receptor content in both myocardium and aorta; however, differences were significant only for abdominal aorta (p < 0.05). Estradiol- 170 treatment caused a tenfold increase in uterine cytoplasmic fraction progesterone receptor content in treated as compared to oophorectomized control females, suggesting that baboon cardiovasculature is less sensitive to changes in endogenousestrogen concentration than is uterus. The ability of estradiol-170 to affect apparent intracellular distribution of baboon cardiovascular estrogen receptors and to elevate cytoplasmic fraction progesterone receptor content suggests that these estrogen receptors are physiologically functional and indicates that estrogen may directly regulate primate cardiovascular cell function.

Hormone receptors of the baboon cardiovascular system. Biochemical characterization of aortic and myocardial cytoplasmic progesterone receptors.

We used the synthetic progestin R5020 (17α,21-dimethyl-19-norpregna-4,9-diene- 3,20-dione) to characterize cytoplasmic progesterone receptors in baboon(Pupio sp.) aorta and myocardium. The relative ability of selected steroids to inhibit binding of radiolabeled R5020 to aortic cytoplasmic progesterone receptors was radioinert R5020, 1.0; progesterone, 0.31; triamcino- lone acetonide, 0.06; testosterone, 0.002; estradiol-17β, 0.01; and cortisol, 0.001. The relative ability of these same steroids to inhibit binding of radiolabeled R5020 to myocardial cytoplasmic progesterone receptors was, respectively, 1.0, 0.58, 0.21, 0.01, 0.01, and 0.001. Both aortic and myocardial cytoplasmic progesterone receptors migrated as macromolecules with a sedimentation coefficient of 8–9S on low ionic strength linear sucrose gradients. Cytoplasmic binding of R5020 was inactivated by incubation at 37°C. Saturation analysis at 2°C showed aorta and myocardium, respectively, contained 41.6 ± 16.1 (mean ± SD) and 14.0 ± 2.8 fmol R5020 binding sites/mg cytosol protein. The dissociation constant for R5020 was 2.8 ± 1.2 nm, aorta, and 2.0 ± 1.1 nM, myocardium. The presence of progesterone receptors in baboon cardiovascular tissues suggests that progestins may directly influence cardiovascular tissue function.

Regulation of ribonucleotide reductase in mammalian cells by chemotherapeutic agents

The reductive conversion of ribonucleotides to deoxyribonucleotides is a prime target for the development of a cancer chemotherapeutic agent. A new series of inhibitors based on the polyhydroxy or polyamino aromatic ring has been developed. These compounds are effective ribonucleotide reductase inhibitors and possess antitumor activity if there are adjacent hydroxy or amino groups. The most effective enzyme inhibitor, 2,3,4-trihydroxybenzohydroxamic acid, is 145 times more effective than hydroxyurea. However, the best antileukemic compound is 3,4-dihydroxybenzohydroxamic acid, which increased the life span of L1210 leukemic mice more than 100%. Structure-activity studies have revealed that the hydroxamic moiety is not essential for activity. The polyhydroxybenzene derivatives reduced the pool sizes of all four deoxynucleotides. This contrasts with the action of hydroxyurea, which depletes only the deoxypurines. The mechanism of inhibition by this group of compounds appears to be related to their ability to trap free radicals, since there is good correspondence between reductase inhibition and free radical destruction. Some naturally occurring cytotoxic and neurological agents which have antineoplastic activity, dopa analogs and phenolic compounds isolated from mushrooms, bear a structural similarity to the new reductase inhibitors. We found that the dopa analogs were also inhibitory to ribonucleotide reductase. Another consequence of polyhydroxybenzoic acid derivative treatment is elevated reductase levels in the cell. Other cell cycle inhibitors that block from late G1 through early G2 also cause an enhanced level of ribonucleotide reductase. However, agents that block in early or mid-G1 or mid or late G2 and mitosis produce lower reductase levels. These data suggest that reductase synthesis is initiated at the G1/S transition point and this enhanced level of activity continues until late S or G2.

Nano-bio-chip sensor platform for examination of oral exfoliative cytology

Oral cancer is a deadly and disfiguring disease that could greatly benefit from new diagnostic approaches enabling early detection. In this pilot study, we describe a nano-bio-chip (NBC) sensor technique for analysis of oral cancer biomarkers in exfoliative cytology specimens, targeting both biochemical and morphologic changes associated with early oral tumorigenesis. Here, oral lesions from 41 dental patients, along with normal epithelium from 11 healthy volunteers, were sampled using a noninvasive brush biopsy technique. Specimens were enriched, immunolabeled, and imaged in the NBC sensor according to previously established assays for the epidermal growth factor receptor (EGFR) biomarker and cytomorphometry. A total of 51 measurement parameters were extracted using custom image analysis macros, including EGFR labeling intensity, cell and nuclear size, and the nuclear-to-cytoplasmic ratio. Four key parameters were significantly elevated in both dysplastic and malignant lesions relative to healthy oral epithelium, including the nuclear area and diameter (P < 0.0001), the nuclear-to-cytoplasmic ratio (P < 0.0001), and EGFR biomarker expression (P < 0.03). Further examination using logistic regression and receiver operating characteristic curve analyses identified morphologic features as the best predictors of disease (area under the curve ≤0.93) individually, whereas a combination of all features further enhanced discrimination of oral cancer and precancerous conditions (area under the curve, 0.94) with high sensitivity and specificity. Further clinical trials are necessary to validate the regression model and evaluate other potential biomarkers, but this pilot study supports the NBC sensor technique as a promising new diagnostic tool for early detection of oral cancer, which could enhance patient care and survival. Cancer Prev Res; 3(4); 518–28. ©2010 AACR.

Alteration in Salivary Function in Early HIV Infection

The etiology of salivary gland hypofunction in HIV(+) patients is unclear. This study was designed to determine the effect of early-stage HIV(+) infection (CD4+ > 200 cells/μL; n = 139) on salivary gland function and the relationship of this dysfunction to the taking of xerostomic medications. Salivary flow rates and the content of electrolytes and antimicrobial proteins in stimulated parotid and submandibular/sublingual saliva were determined. Compared with healthy controls (n = 50), the HIV(+) group showed significant reductions in flow rates of unstimulated whole (35%), stimulated parotid (47%), unstimulated submandibular/sublingual (23%), and stimulated submandibular/sublingual (39%) saliva. The flow rates for the HIV(+) patients taking xerostomic medications did not differ from those of patients who did not. Concentrations of some salivary gland components were altered in the HIV(+) group. Analysis of these data suggests that salivary gland function is adversely affected early in HIV infection and that these changes do not appear to be compounded by the taking of xerostomic medications.

Measuring short-term γ-irradiation effects on mouse salivary gland function using a new saliva collection device

A restraining device was designed specifically for the collection of whole saliva from mice without using anesthesia. As the procedure does not involve surgical cannulation of the salivary glands, saliva can be collected from the same mouse at different times. The time between the injection of a secretory stimulant (pilocarpine) and the appearance of saliva in the mouth (lag time) was 100.5 +/-8.5 s (mean+/-S.E.M., n=10) for control mice. The volume of saliva collected in the first 5 min was three times greater than that collected between 15 and 20 min. The average flow rate for a collection period of 15 min was 16.7 +/-1.8 microl/min (n=10). The flow rate was decreased 50% (P<0.005) whereas the lag time was increased more than 300% (P<0.05) at 24 h after irradiation. The concentrations of a 23.5-kDa protein and a mucin were decreased after irradiation whereas there was no significant effect on the concentration of amylase or peroxidase.

Hormone receptors of the baboon cardiovascular system. Biochemical characterization of myocardial cytoplasmic androgen receptors

Using the synthetic androtfen R1881 (17β-hydroxy-17α-methyl-estra-4,9,ll-trien-3-one) as probe, we identified cytoplasmic androgen receptors in baboon myocardium. The relative binding affinity of selected steroids for the androgen receptor was R1881, 100%; 5α-dihydrotestosterone, 32.8%; testosterone, 29.6%, progesterone, 7.2%; R5020, 1.0%; and estradiol-17β, 5.8%. The androgen receptor migrated on low ionic strength linear sucrose density gradients as a macromolecule with a sedimentation coefficient of 8.5S. Saturation analysis performed at 2°C (available sites) showed that the androgen receptor content of baboon myocardial cytoplasmic extracts was 5.9 ± 1.4 μmol/mg protein and that the dissociation constant for R1681 was 1.16 ± 0.30 run. These cytoplasmic androgen receptors are indicated to be physiologically functional by previous autoradiographic studies (McGill et aL, 1980; McGill and Sheridan, 1981) that showed localization of radloisotope In nuclei of myocardial fibers following injection of baboons with 5α-dihydrotestosterone. Circ Res 49: 1010–1016, 1881

Role for notch signaling in salivary acinar cell growth and differentiation

The Notch pathway is crucial for stem/progenitor cell maintenance, growth and differentiation in a variety of tissues. The Notch signaling is essential for Drosophila salivary gland development but its role in mammalian salivary gland remains unclear. The human salivary epithelial cell line, HSG, was studied to determine the role of Notch signaling in salivary epithelial cell differentiation. HSG expressed Notch 1 to 4, and the Notch ligands Jagged 1 and 2 and Delta 1. Treatment of HSG cells with inhibitors of γ‐secretase, which is required for Notch cleavage and activation, blocked vimentin and cystatin S expression, an indicator of HSG differentiation. HSG differentiation was also associated with Notch downstream signal Hes‐1 expression, and Hes‐1 expression was inhibited by γ‐secretase inhibitors. siRNA corresponding to Notch 1 to 4 was used to show that silencing of all four Notch receptors was required to inhibit HSG differentiation. Normal human submandibular gland expressed Notch 1 to 4, Jagged 1 and 2, and Delta 1, with nuclear localization indicating Notch signaling in vivo. Hes‐1 was also expressed in the human tissue, with staining predominantly in the ductal cells. In salivary tissue from rats undergoing and recovering from ductal obstruction, we found that Notch receptors and ligands were expressed in the nucleus of the regenerating epithelial cells. Taken together, these data suggest that Notch signaling is critical for normal salivary gland cell growth and differentiation. Developmental Dynamics 238:724–731, 2009.

Sexual dimorphism characterizes baboon myocardial androgen receptors but not myocardial estrogen and progesterone receptors

Using biochemical methods we established that estrogen receptor content and distribution and progesterone receptor content in female and male baboon myocardium did not differ between sexes. In contrast, myocardial androgen receptor distribution between cytosolic and nuclear compartments was sexually dimorphic. Female baboon myocardial androgen receptors were restricted to the cytosolic compartment, whereas male myocardial androgen receptors were distributed between the cytosolic and nuclear compartments. Using human estrogen receptor cDNA we showed that baboon aorta, myocardium and uterus contain a 6.3 kb estrogen receptor transcript. Analyses performed with human progesterone receptor cDNA established that baboon aorta and uterus contain an 8 kb progesterone receptor transcript; however, progesterone receptor transcripts were not demonstrable in baboon myocardial RNA preparations. Because relative hybridization signal intensity reflected known uterine and aortic progesterone receptor content, failure to detect progesterone receptor transcripts in myocardial preparations may reflect sensitivity limitations and the fact that aortic progesterone receptor content is 5-fold greater than that of myocardium. Immunocytochemical analyses demonstrated that baboon myocardial progesterone receptors were present in greater than 25% of myocytes and generally absent from other myocardial cells. Our studies establish that: (1) gonadal steroid hormone receptor gene transcription occurs in cells of the baboon cardiovasculature, (2) these steroid hormone receptors may be physiologically functional, and (3) gonadal steroid hormone receptors may be restricted to specialized cells of the cardiovasculature.

Distinct pathways of ERK activation by the muscarinic agonists pilocarpine and carbachol in a human salivary cell line.

Cholinergic-muscarinic receptor agonists are used to alleviate mouth dryness, although the cellular signals mediating the actions of these agents on salivary glands have not been identified. We examined the activation of ERK1/2 by two muscarinic agonists, pilocarpine and carbachol, in a human salivary cell line (HSY). Immunoblot analysis revealed that both agonists induced transient activation of ERK1/2. Whereas pilocarpine induced phosphorylation of the epidermal growth factor (EGF) receptor, carbachol did not. Moreover, ERK activation by pilocarpine, but not carbachol, was abolished by the EGF receptor inhibitor AG-1478. Downregulation of PKC by prolonged treatment of cells with the phorbol ester PMA diminished carbachol-induced ERK phosphorylation but had no effect on pilocarpine responsiveness. Depletion of intracellular Ca2+ ([Ca2+]i by EGTA did not affect ERK activation by either agent. In contrast to carbachol, pilocarpine did not elicit [Ca2+]i mobilization in HSY cells. Treatment of cells with the muscarinic receptor subtype 3 (M3) antagonist N-(3-chloropropyl)-4-piperidnyl diphenylacetate decreased ERK responsiveness to both agents, whereas the subtype 1 (M1) antagonist pirenzepine reduced only the carbachol response. Stimulation of ERKs by pilocarpine was also decreased by M3, but not M1, receptor small interfering RNA. The Src inhibitor PP2 blocked pilocarpine-induced ERK activation and EGF receptor phosphorylation, without affecting ERK activation by carbachol. Our results demonstrate that the actions of pilocarpine and carbachol in salivary cells are mediated through two distinct signaling mechanisms-pilocarpine acting via M3 receptors and Src-dependent transactivation of EGF receptors, and carbachol via M1/M3 receptors and PKC-converging on the ERK pathway.