Higher order genomic organization and epigenetic control maintain cellular identity and prevent breast cancer.

Cells establish and sustain structural and functional integrity of the genome to support cellular identity and prevent malignant transformation. In this review, we present a strategic overview of epigenetic regulatory mechanisms including histone modifications and higher order chromatin organization (HCO) that are perturbed in breast cancer onset and progression. Implications for dysfunctions that occur in hormone regulation, cell cycle control, and mitotic bookmarking in breast cancer are considered, with an emphasis on epithelial-to-mesenchymal transition and cancer stem cell activities. The architectural organization of regulatory machinery is addressed within the contexts of translating cancer-compromised genomic organization to advances in breast cancer risk assessment, diagnosis, prognosis, and identification of novel therapeutic targets with high specificity and minimal off target effects.

Pain and depression predict self-reported fatigue/energy in lupus.

UNLABELLED: This study examined the contribution of pain and psychological distress to fatigue. METHODS: One-hundred and twenty-five adult Caucasian and Hispanic lupus patients participated in this study. Demographic data, patient- and physician-reported disease activity, as well as psychological functioning, were collected. Fatigue, pain, and vitality were measured using visual analogue scales as well as a subscale of the SF-36 questionnaire. Linear and hierarchical regression analyses were conducted. In the regression analysis, ethnicity was entered at the first step, followed by age, income and education at step 2, pain and disease activity measures at step 3, and psychological measurements at step 4. RESULTS: In the linear regression analysis, Caucasians reported more fatigue. Fatigue positively correlated with income, education, pain, patient-reported disease activity, helplessness, and depression, and negatively with internality, and the energy analysis mirrored the results of the fatigue analysis. In the first regression analysis, fatigue was the dependent variable. At step 1, Caucasians reported more fatigue. At step 2, no other demographic variables were significant. At step 3, pain and disease activity measures were significant when entered as a block; however, pain independently explained a large amount of variance. At step 4, psychological factors were significant as a block, with depression being the strongest predictor. In the second analysis, energy was the dependent variable. At step 1, Hispanics reported more energy. At step 2, demographic variables were not significant. At step 3, pain and disease activity were significant when entered as a block; however, only pain uniquely predicted energy. At step 4, psychological factors were significant as a block, with depression as the major contributor. CONCLUSIONS: Both pain and depression were found to be strong predictors of fatigue, and negatively correlated with energy. Disease activity did not appear to play a significant role in lupus fatigue. These findings support the importance of managing depression and pain in order to reduce fatigue in patients with systemic lupus erythematosus.

Depression predicts self-reported disease activity in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease that can significantly impact both physiological and psychological functioning. In order to examine the relationship between psychological functioning and disease activity in SLE, we administered instruments that collected sociodemographic information and measured indices of disease activity and psychosocial functioning from 125 adult Hispanic and White patients with SLE. Patients were recruited from four healthcare settings in the greater Southern California area. Both cross-sectional and longitudinal relationships between depression and disease activity were evaluated. Cross-sectional findings revealed that depression and ethnicity were independently correlated with self-reported disease activity. Longitudinally, depression alone predicted self-reported disease activity. These data suggest that depression may play a significant role in the health status of SLE patients and serve as an important target for clinical intervention.

The Patient Reported Outcomes in Lupus (PATROL) study: role of depression in health-related quality of life in a Southern California lupus cohort.

UNLABELLED: This study examines the relationship between psychosocial factors, ethnicity, disease activity and quality of life in systemic lupus erythematosus. METHODS: One hundred and twenty-five adult Caucasian and Hispanic lupus patients were recruited from four Southern California medical centers. Linear regression analysis was performed to assess the correlation of ethnicity, socioeconomic factors (age, income), and disease activity (patient and physician reported), as well as psychological (depression, internality, helplessness) variables with quality of life (QOL) as measured by the Short Form (SF)-36. Hierarchical multiple regression analysis was then used to determine the stepwise contribution of the above determinants on the eight domains of the SF-36 questionnaire. RESULTS: Depression negatively correlated with QOL in both Caucasians (r -0.488 to -0.660) and Hispanics (r -0.456 to -0.723). Patient-reported disease activity was moderately related (r -0.456 to -0.698) to seven of the eight SF-36 domains in Hispanics, and none in Caucasians. Physician-reported disease activity, measured by SLEDAI, did not correlate with QOL among Hispanics or Caucasians. When linear and hierarchical regression was used, depression significantly correlated (p < 0.0001) with the majority of the SF-36 domains, except general health, while age had a significant effect in only one domain of the SF-36, physical functioning (p < 0.0001). CONCLUSION: Depression, and not disease activity, appears to have a major influence on quality of life in both Hispanic and Caucasian patients in this lupus cohort.

Starvation and hypothyroidism exert an overlapping influence on rat hepatic messenger RNA activity profiles.

To assess the effect of starvation and to explore the potential interrelationship of starvation and thyroid status at the pretranslational level, we have analyzed by two-dimensional gel electrophoresis, the hepatic translational products of starved and fed euthyroid and hypothyroid rats. 5 d of starvation resulted in a statistically significant change in 27 of 240 products visualized, whereas hypothyroidism caused a change in 20, both in comparison with the fed euthyroid state. Of considerable interest was that 68% of all changing messenger (m)RNA sequences were common to the hypothyroid and starved groups and showed the same directional shift. Further, both starvation and hypothyroidism yielded comparable decreases in total hepatic cytoplasmic RNA content. Although it has been well established that the level of circulating triiodothyronine (T3) and the level of hepatic nuclear receptors fall in starvation, this reduction cannot account for the observed decrease of total hepatic RNA nor for all of the alterations in the concentrations of specific mRNA sequences. Thus, administration of T3 to starved animals in a dose designed to occupy all nuclear T3 receptors fails to prevent the fall in total RNA and the majority of starvation-induced changes in the level of mRNA sequences. Moreover, starvation of athyreotic animals results in a further decrease in total RNA and in a further change in the level of individual mRNA species. We conclude, therefore, that although the reduced levels of circulating T3 and the nuclear T3 receptors can contribute to the observed results of starvation, the starvation-induced changes are not exclusively mediated by this factor. The striking overlap in the genomic response between hypothyroid and starved animals raises the possibility that those biochemical mechanisms regulated at a pretranslational level by T3 are either not helpful or injurious to the starving animal. The reduction in circulating T3 and nuclear receptor sites together with T3-independent mechanisms initiated in the starved animal may constitute redundant processes designed to conserve energy and substrate in the nutritionally deprived organism.

Hepatic messenger ribonucleic acid activity profiles in experimental azotemia in the rat. Relationship to food intake and thyroid function.

We have studied the hepatic messenger RNA (mRNA) activity profile in chronically azotemic rats and sought to determine whether the observed changes could be mediated either by reduced food intake or diminished thyroid function at the tissue level. mRNA activity profiles were produced by two-dimensional gel electrophoretic separation of radioactively labeled products of an in vitro reticulocyte lysate system which had been programmed by hepatic RNA. Of the approximately 240 translational products identified in this system, seven sequences were consistently altered in azotemia. In pair-fed animals six of these also decreased, but the alterations in three were depressed to a significantly lesser extent in the pair-fed group. Moreover, analysis of covariance suggested that food intake could account for the differences in only one sequence. The possibility that the mRNA activity profile in azotemia could represent the effects of diminished thyroid function was minimized by the finding that the reductions in plasma thyroxine (T4) and triiodothyronine (T3) levels observed were due largely to reduced plasma protein binding, with maintenance of the mean free T4 and free T3 concentrations within the normal range. The changes in only one mRNA sequence could be related to free T3 levels alone. Our findings, therefore, indicate that although diminished food intake and reduced thyroid function may contribute to some of the observed changes in the mRNA activity profiles, the bulk of alterations in azotemia appear to be mediated by other mechanisms. The striking overlap between the sequences affected by azotemia and pair-feeding raises the speculation that altered gene expression in azotemia may reflect an impaired hepatic response at the pretranslational level to metabolic signals associated with food intake.

Construction, production, and characterization of humanized anti-Lewis Y monoclonal antibody 3S193 for targeted immunotherapy of solid tumors.

The Lewis Y (Ley) antigen is a blood group-related antigen that is expressed in a high proportion of epithelial cancers (including breast, colon, ovary, and lung cancer) and is an attractive target for monoclonal antibody-directed therapy. The murine monoclonal 3S193 (IgG3) was generated in BALB/c mice by immunization with Ley-expressing cells of the MCF-7 breast carcinoma cell-line. The murine 3S193 showed high specificity for Ley in ELISA tests with synthetic Ley and Ley-containing glycoproteins and glycolipids and also reacted strongly in rosetting assays and cytotoxic tests with Ley-expressing cells. We generated a humanized form of the murine 3S193 antibody by linking cDNA sequences encoding the variable region of murine 3S913 with frameworks of the human KOL heavy chain and REI K chain. The genes for the humanized 3S193 monoclonal antibody IgG1 were transfected into mouse myeloma NS0 cells and cloned for the establishment of high antibody-producing colonies. Humanized 3S193 antibody was subsequently produced through in vitro culture and under good manufacturing practice conditions using hollow-fiber bioreactors. The purified humanized 3S193 (hu3S193) was subsequently characterized and validated for use in preliminary immunotherapy investigations. hu3S193 reacted specifically with Ley antigen, with similar avidity to the murine form. hu3S193 demonstrated potent immune effector function, with higher antibody-dependent cell-mediated cytotoxicity than its murine counterpart and potent complement-dependent cytotoxicity (ED50, 1.0 microg/ml). The in vivo immunotherapeutic potential of hu3S193 was assessed in a human breast xenograft model using MCF-7, Ley-positive cells. Six i.v. doses of up to 1 mg of hu3S193 were administered to animals bearing established tumors (120-130 mm3) with no significant effect on tumor growth. In contrast, in an MCF-7 xenograft preventive model, a 1-mg hu3S193 dosage schedule was able to significantly slow tumor growth compared with placebo and isotype-matched control IgG1 antibody. hu3S193 has promise for immunotherapy of Ley-positive tumors and is currently entering Phase I clinical trials.

Idiotope determining regions of a mouse monoclonal antibody and its humanized versions. Identification of framework residues that affect idiotype expression.

The contribution of framework regions (FRs) of antibody-variable domains to idiotype expression was studied by examining the interaction of various "humanized" versions of a mouse anti-TNF alpha monoclonal antibody (mAb78) with polyclonal and two monoclonal antibodies (mAb1G3 and mAb9F1), generated against the mAb78 idiotype. Humanized mAb78, bearing human constant domains and mouse complementarity-determining regions (CDRs) inserted with human FRs, was found to be five to sevenfold less reactive than mAb78 with polyclonal anti-idiotype antibodies and 200 to 300-fold less active in neutralizing TNF alpha. The substitution of heavy-chain FRs residues of the humanized antibody with original mouse residues 28 to 30, 48 to 49, 67 to 68, 70 to 71, 78, 80 and 82 progressively restored the immunoreactivity with polyclonal immunoglobulin Gs to the level of a version having mouse heavy chain and human light chain FRs, and increased 10 to 20-fold the TNF alpha neutralizing activity. This suggests that at least some of these residues are critical for TNF alpha binding as well as for the expression of idiotopes that are strongly immunogenic in syngeneic animals. All antibody versions with either human or mouse FRs were able to bind to various extents mAb1G3, a gamma-type anti-Id antibody that inhibits mAb78/TNF alpha interaction by paratope blockade. At variance, only the antibody versions containing mouse FRs were able to bind mAb9F1, an alpha-type anti-Id antibody unable to block the access of TNF alpha to mAb78 paratopes. Substitution of heavy chain FR residues 28 to 30 markedly decreased the binding of mAb1G3 (100 to 1000-fold). This suggests that these antibodies recognize CDR and FR idiotopes, respectively, that can be drastically modified by changes in the FRs. In conclusion, the results suggest that CDRs as well as FRs markedly contribute to antibody Id expression. Although strongly immunogenic idiotopes are probably located within the CDRs, the results also suggest that some FR residues are critically involved in shaping antibody Id diversity by affecting the structure of CDR-related idiotopes.

Differential thyroid hormone-regulated rat thyrotropin beta gene expression detected by blot hybridization.

In the rat, there is a single TSH beta-subunit gene represented by three exons interrupted by two introns. This gene contains two promoters which determines the synthesis of two mRNAs with 5'-untranslated regions that differ by 43 base pairs. This study evaluates the steady state levels of these TSH beta mRNAs in various thyroidal states. Blot hybridization analyses of pituitary mRNA with synthetic probes designed to detect either one or both TSH beta mRNAs were performed. One probe corresponds to 24 bases in the 5'-untranslated region of mRNA1 and a second corresponds to 25 nucleotides in the coding region and detects both mRNA1 and mRNA2. These studies indicate the presence of TSH beta mRNA species of indistinguishable size consistent with the presence of two TSH beta mRNAs that contain slightly different 5'-untranslated regions. Comparison of pituitary RNA obtained from normal and hypothyroid rats reveals that the shorter mRNA (mRNA2) is increased approximately 6- to 8-fold with hypothyroidism while the abundance of the longer mRNA (mRNA1) is relatively unchanged. Treatment of either normal or hypothyroid animals with T3 decreases the abundance of mRNA2 while again mRNA1 is relatively unaffected. Thus, although both mRNAs are detected, only one mRNA is dramatically altered by thyroidal status. Therefore, the single rat TSH beta gene is transcribed into two mRNAs via the use of alternative promoters of which only one is markedly regulated by thyroid hormones.

Thyroid hormones regulate rat thyrotropin beta gene promoter activity expressed in GH3 cells.

Thyroid hormones suppress the synthesis of TSH in part by decreasing the rate of alpha and TSH beta gene transcription. Cis-acting DNA sequences present in the rat TSH beta subunit gene that are induced in transcriptional regulation by thyroid hormone have been identified by deletion-mutation and transient expression studies. Plasmid expression vectors were constructed including 2900, 900, 204, 77, 17 base pairs (bp) of 5'-flanking sequence and exon (5'-untranslated sequence, transcriptional start sites) fused to the coding region of the bacterial chloramphenicol acetyltransferase (CAT) gene. The transfected chimaeric plasmids demonstrated expression (with TSH beta DNA sequences in the 5'- to -3'-but not 3'- to -5'-orientation) in both a clonal pituitary cell line, GH3, and primary pituitary cell cultures, both of which are responsive to thyroid hormones. T3 (10(-11) M to 10(-7) M) treatment of transfected cells produced a dose-dependent decrease in CAT expression with a maximal 70% decrease at 10(-8) M. While a decrease in the basal level of expression was noted with progressive removal of both 5'-flanking and intronic sequences adjacent to exon 1, the fold-decrease in response to T3 was equivalent even in the 57 bp construct. In contrast, T3 had no effect on CAT expression directed by the promoter of the herpes simplex virus thymidine kinase gene. Thus, the rat TSH beta gene 5'-flanking region can direct heterologous gene expression in GH3 cells and contains sequences which have properties of a putative cis-active T3 responsive regulatory element(s).2+he

Thyrotropin-releasing hormone stimulates the activity of the rat thyrotropin beta-subunit gene promoter transfected into pituitary cells.

In order to investigate the molecular mechanism(s) by which TRH regulates the biosynthesis of TSH, we are studying the effects of TRH on the expression of the TSH subunit genes (alpha and TSH beta). To study the structure-function relation of TRH stimulation of the activity of the single rat TSH beta gene, chimaeric plasmids were constructed. The 5'-flanking region of the rat TSH beta gene including exon 1 (5'-untranslated region) was inserted into a promoterless, modified pBR, chloramphenicol acetyltransferase (CAT) expression vector. After transfection, specific TSH beta promoter activity was evident in both TRH-responsive pituitary-derived GH3 and primary pituitary cell cultures. To determine potential regulation of TSH beta promoter-directed activity in these cells by TRH, cells were incubated with media containing TRH (10(-7) to 10(-11) M) for 1 to 48 h. TRH stimulated a 1.5- to 3-fold increase in TSH beta promoter activity. Concomitant with an increase in CAT activity was an anticipated increase in PRL synthesis in the GH3 cells in response to TRH. The TRH effect on the TSH beta gene was specific; no increase in CAT activity was detected for TKCAT (thymidine kinase of herpes simplex virus promoter), pBRCAT (no promoter), or TSH beta CAT (3'-5'-orientation). Similar results were obtained using primary pituitary cell cultures. Deletion mutation analysis indicated that TRH sensitivity was detected in a 1.1 kilobase, but not in a 0.38 kilobase TSH beta gene fragment suggesting that the TRH responsive element(s) resides at least in part within the 700 base pairs of the 5'-flanking sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of the thyroid hormone-responsive messenger RNA spot 11 as apolipoprotein-A1 messenger RNA and effects of the hormone on the promoter.

The induction of rat hepatic mRNA S11 by L-T3 (T3) is a useful model for studying the mechanisms of thyroid hormone action. Although numerous reports have examined the response of mRNA S11 to various physiological and hormonal manipulations, the role of S11 protein in cellular metabolism remains unknown. In this study we show that mRNA S11 is abundantly expressed and regulated by T3 only in liver and small intestine. High levels of the mRNA are present at birth, but drop sharply between 30-60 days of age. These and other features of the S11 gene product were similar to those of rat apolipoprotein-A1 (Apo-A1). The sequence of S11 cDNA was identical to a portion of the Apo-A1 mRNA, thus confirming identity of the S11 mRNA. To examine whether DNA sequences immediately adjacent to the transcription start site mediate the effects of thyroid hormone, we measured the activity of an Apo-A1 gene fragment, U-1 (-474 to -7) using a transient transfection assay. The activity of the full-length U-1 DNA in HuH-7 hepatoma cells was 2- to 2.5-fold higher in the presence of thyroid hormone. This finding closely matched previous results using the in vitro nuclear run-on assay. Internal deletion of a motif that resembles a thyroid hormone response element from U-1 DNA not only abolished the induction by T3, but suppressed promoter activity by 3- to 4-fold in response to the hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

Altered Na+-K+ pump activity and plasma lipids in salt-hypertensive Dahl rats: relationship to Atp1a1 gene.

A genetic variant of the gene for the alpha(1)-isoform of Na(+)-K(+)-ATPase (Atp1a1) was suggested to be involved in the pathogenesis of salt hypertension in Dahl rats through altered Na(+):K(+) coupling ratio. We studied Na(+)-K(+) pump activity in erythrocytes of Dahl salt-sensitive (SS/Jr) rats in relation to plasma lipids and blood pressure (BP) and the linkage of polymorphic microsatellite marker D2Arb18 (located within intron 1 and exon 2 of Atp1a1 gene) with various phenotypes in 130 SS/Jr x SR/Jr F(2) rats. Salt-hypertensive SS/Jr rats had higher erythrocyte Na(+) content, enhanced ouabain-sensitive (OS) Na(+) and Rb(+) transport, and higher Na(+):Rb(+) coupling ratio of the Na(+)-K(+) pump. BP of F(2) hybrids correlated with erythrocyte Na(+) content, OS Na(+) extrusion, and OS Na(+):Rb(+) coupling ratio, but not with OS Rb(+) uptake. In F(2) hybrids there was a significant association indicating suggestive linkage (P < 0.005, LOD score 2.5) of an intragenic marker D2Arb18 with pulse pressure but not with mean arterial pressure or any parameter of Na(+)-K(+) pump activity (including its Na(+):Rb(+) coupling ratio). In contrast, plasma cholesterol, which was elevated in salt-hypertensive Dahl rats and which correlated with BP in F(2) hybrids, was also positively associated with OS Na(+) extrusion. The abnormal Na(+):K(+) stoichiometry of the Na(+)-K(+) pump is a consequence of elevated erythrocyte Na(+) content and suppressed OS Rb(+):K(+) exchange. In conclusion, abnormal cholesterol metabolism but not the Atp1a1 gene locus might represent an important factor for both high BP and altered Na(+)-K(+) pump function in salt-hypertensive Dahl rats.

Cell-to-cell movement of potexviruses: evidence for a ribonucleoprotein complex involving the coat protein and first triple gene block protein.

The triple gene block proteins (TGBp1-3) and coat protein (CP) of potexviruses are required for cell-to-cell movement. Separate models have been proposed for intercellular movement of two of these viruses, transport of intact virions, or a ribonucleoprotein complex (RNP) comprising genomic RNA, TGBp1, and the CP. At issue therefore, is the form(s) in which RNA transport occurs and the roles of TGBp1-3 and the CP in movement. Evidence is presented that, based on microprojectile bombardment studies, TGBp1 and the CP, but not TGBp2 or TGBp3, are co-translocated between cells with viral RNA. In addition, cell-to-cell movement and encapsidation functions of the CP were shown to be separable, and the rate-limiting factor of potexvirus movement was shown not to be virion accumulation, but rather, the presence of TGBp1-3 and the CP in the infected cell. These findings are consistent with a common mode of transport for potexviruses, involving a non-virion RNP, and show that TGBp1 is the movement protein, whereas TGBp2 and TGBp3 are either involved in intracellular transport or interact with the cellular machinery/docking sites at the plasmodesmata.

Absence of detectable chorionic gonadotropin subunit messenger ribonucleic acids in the rat placenta throughout gestation.

We hypothesized that if a CG similar in structure to other glycoprotein hormones was present in the rat placenta, then mRNAs encoding its subunits would be detectable. To investigate the possible presence of a CG in the rat, we attempted to detect mRNAs encoding the alpha- and CG beta-like subunits in the placentae of timed pregnant rats by sensitive blot hybridization analyses using cloned cDNAs encoding alpha- and beta-subunits of LH derived from rat pituitary mRNA. No subunit-specific mRNAs in placentae of pregnant rats at 4-21 days gestation were detected at either high or low stringencies of hybridization. However, under similar conditions, subunit-specific mRNAs were readily observed in total pituitary RNA of normal as well as ovariectomized rats. Moreover, hybridization with a cDNA for alpha-tubulin, a major component of the cytoskeleton, yielded easily detectable bands in rat placental RNA. In addition, hybridization analysis, under low stringency conditions, of restriction enzyme digests of rat genomic DNA with a rat LH beta-cDNA, which would detect LH beta subunit-like genes, suggests the presence of a single gene that, in fact, encodes the rat LH beta-subunit. We conclude that mRNAs encoding for proteins structurally homologous to rat LH subunits are absent in the rat placenta and that only a single LH beta-like gene is present in the rat. The luteotropic activity associated with the rat placenta must be related to a gonadotropin-like hormone whose structure is dissimilar from that of CG.

A cryptic peptide from the preprothyrotropin-releasing hormone precursor stimulates thyrotropin gene expression.

The precursor peptide of TRH (prepro-TRH) contains five copes of pro-TRH linked by other peptide sequences. These peptides are coprocessed with TRH in the median eminence of the hypothalamus and released into the portal circulation, rendering this family of peptides available to act at the level of the anterior pituitary. Therefore, we tested the potential bioactivity of one cryptic peptide, prepro-TRH amino acids 160-169 [prepro-TRH-(160-169)], in a TRH-responsive pituitary cell line (GH3). In a heterologous TSH expression assay, we found that prepro-TRH-(160-169) stimulated TSH beta gene promoter activity in a time- and dose-dependent manner; moreover, the effect of prepro-TRH-(160-169) was more rapid and of greater magnitude than that of TRH on TSH beta-directed chloramphenicol acetyltransferase synthesis. In the same cells, we found that prepro-TRH-(160-169) stimulated PRL synthesis and secretion, but the effect was similar in magnitude and duration to that of TRH. The effect of prepro-TRH-(160-169) appears to be additive to that of TRH, suggesting that prepro-TRH-(160-169) may act through a mechanism separate from that of TRH. Thus, prepro-TRH-(160-169) has potent endocrinological effects at the level of the genome.

Distribution of thyroid hormone-responsive translated products in rat liver polysome and postribosomal ribonucleoprotein populations.

We investigated the possibility that hypothyroidism, like starvation, leads to a major disaggregation of polyribosomes and redistribution of mRNA to the nonpolysomal mRNA particle (mRNP) pool. The analysis involved the use of a translational assay of hepatic RNA obtained from bound and free polysomes and the subribosomal fractions (mRNP) of euthyroid and hypothyroid rats. Two-dimensional gel electrophoretograms of the in vitro synthesized 35S-labeled proteins revealed distinct protein patterns associated with each subcellular compartment. Of the 18 quantified T3-responsive sequences, the activities of 7 were preferentially associated with bound polysomes, 9 with free polysomes, and 2 with subribosomal mRNP in euthyroid rat livers. No major redistribution occurred in the transition from the euthyroid to the hypothyroid state. The distribution of mRNA activity of 1 T3-responsive sequence, that coding for Spot 14 (mRNAS14), was compared with the distribution of hybridizable mRNA, as determined with the use of a specific cDNA probe. In the euthyroid state, both techniques showed that approximately 70% of the total hepatic mRNAS14 was associated with free polysomes and 30% was associated with subribosomal complexes. A similar distribution was characteristic of the hypothyroid state. These findings demonstrate that T3-responsive sequences are found in all three mRNA compartments and that thyroid hormone action is not typically associated with a redistribution of mRNA among subcellular pools.

Rapid simultaneous measurement of rat alpha- and thyrotropin (TSH) beta-subunit messenger ribonucleic acids (mRNAs) by solution hybridization: regulation of TSH subunit mRNAs by thyroid hormones.

Using a sensitive method for the simultaneous measurement of TSH subunit mRNAs, we have investigated their hormonal regulation in the pituitary glands of normal and hypothyroid rats. Oligodeoxyribonucleotides (probes) complementary to coding regions of rat alpha- and TSH beta-subunit mRNAs were synthesized. These probes were 5'-end labeled with gamma-[32P] ATP and hybridized with total pituitary RNA obtained from T3-treated and untreated normal and hypothyroid rats. The samples were then exposed to S1 nuclease to digest single stranded nucleic acids. Specific hybridization of probes to the TSH subunit mRNAs would yield double stranded structures resistant to this enzyme. Measurement of the amount of undigested probes by denaturing polyacrylamide gel electrophoresis, autoradiography, and densitometry permits quantification of these mRNAs. Both rat alpha and TSH beta mRNAs were detected with as little as 0.1 microgram total pituitary RNA, representing a more than 10-fold increase in sensitivity compared to a standard RNA blot hybridization assay. Thyroidectomy resulted in a 3- to 5-fold increase, whereas T3 treatment caused a significant decrease in the subunit mRNAs in both normal and hypothyroid animals. However, in all treatment groups, the TSH beta mRNA was affected to a greater extent than the alpha mRNA by the changes in thyroid status. The ratio of alpha- to beta-subunit mRNAs was decreased with hypothyroidism and increased with T3 treatment. This assay allows simultaneous quantification of multiple mRNAs from a single pituitary gland within 48 h and should facilitate studies of the regulation of mRNAs encoding TSH subunits specifically and other pituitary proteins in general.

Thyrotropin-releasing hormone stimulates c-jun and c-fos messenger ribonucleic acid levels: implications for calcium mobilization and protein kinase-C activation.

The hypothalamic neuropeptide TRH, through G-protein-coupled transmembrane pituitary receptors, rapidly stimulates intracellular signaling events that, in turn, stimulate gene transcription. Our previous studies in transfected pituitary tumor cells indicated that TRH stimulation of thyrotropin beta-subunit (TSH beta) gene expression involves both calcium mobilization and protein kinase-C activation. To characterize the gene-proximal elements of the intracellular signaling pathways involved, we examined the effects of TRH, ionomycin, and phorbol ester (TPA) on cellular protooncogenes (c-jun and c-fos) known to be responsive to calcium mobilization and protein kinase-C activation. TRH stimulated a 3-fold increase in both c-jun and c-fos mRNA levels within 1 h, followed by a rapid decline in steady state mRNA levels. A secondary response to the single administration was noted, culminating in a 5-fold stimulation at 20 h. The increase in c-jun and c-fos mRNA levels occurred before the increased steady state mRNA levels of both PRL and TSH beta chimera in transfected pituitary GH3 cells. Furthermore, we examined the role of calcium in these effects using the ionophore ionomycin to elevate and TMB-8 to decrease intracellular calcium. We used the phorbol ester TPA to investigate the effects of increased protein kinase-C activity and H7 or pretreatment with TPA to monitor the decreased kinase activity. Our data indicate that calcium mobilization and protein kinase activation represent distinct components of the signaling events initiated by TRH resulting in increased c-jun and c-fos mRNA levels. Only c-fos mRNA is increased by all three factors, suggesting that c-fos may be a key element in mediating the intracellular processes reflecting TRH action.

A cryptic peptide (160-169) of thyrotropin-releasing hormone prohormone demonstrates biological activity in vivo and in vitro.

TRH is synthesized as a precursor peptide containing five copies of the sequence Gln-His-Pro-Gly, QHPG, flanked by paired basic amino acids, and linked by other peptides. We tested one cryptic peptide, PPT (160-169, SFPWMESDVT), as a possible physiological regulator of pituitary activity in vivo. Male rats were cannulated (jugular) and received a single dose of either PPT or TRH (10(-8)-10(-6) M). PPT caused no consistent effects on either TSH or PRL secretion, while TRH stimulated the secretion of both hormones. However, PPT stimulated a dose-dependent increase in both pituitary TSH beta and PRL mRNA content at 240 min similar to TRH. In primary cultures of rat pituitaries, PPT stimulated a maximum 4-fold increase in TSH beta mRNA and a 2-fold increase in PRL mRNA in 4 h, while TRH increased both TSH beta and PRL mRNA approximately 3-fold. Again, PPT had no significant effect on TSH or PRL secretion into the medium. Thus, PPT appears to be a physiological regulator of both TSH and PRL synthesis, but, unlike TRH, does not act as a secretagogue.