Long-term storage of information in DNA.
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Archaeology↗
Biomedical subjects
C Bancroft
Publications and source records attributed to C Bancroft.
IL-12- and IL-2-induced tumor regression in a new murine model of oral squamous-cell carcinoma is promoted by expression of the CD80 co-stimulatory molecule and interferon-gamma.
Therapy with IL-12 or IL-2 induces tumor regression in only a few patients with head-and-neck squamous cell carcinoma (SCC), and the factors promoting responsiveness have not been well defined. In this study, we examined whether combined IL-12 and IL-2 therapy can induce tumor regression in a new murine model of oral SCC and determined if the anti-tumor response is promoted by expression of the immune co-stimulatory molecule CD80 and cytokine IFN-gamma. In CD80-positive or -negative subclones of a BALB/c oral SCC line in syngeneic mice, we showed that systemic rIL-12 alone was comparable in effectiveness to combined therapy with IL-12 and peri-tumoral rIL-2, inducing complete regression of the CD80(+) line B7E11-4scid. However, therapy with these cytokines had no effect on growth of the CD80(-) subclone B7E3-4scid and did not induce complete regression of the CD80(+) subclone B7E11-4scid in congenic BALB/c IFN-gamma knockout mice, indicating that expression of the CD80 co-stimulatory molecule and IFN-gamma contributes to tumor regression. In cytokine-treated mice that rejected the CD80(+) SCC line, an increase in infiltrating CD4(+) lymphocytes and apoptotic bodies within the tumor specimens was observed, and resistance to rechallenge with the same tumor was detected in 50% of recipients, consistent with an immune response. Our results provide evidence that regression of oral head-and-neck SCC may be induced by therapy with systemic IL-12 and that expression of the CD80 co-stimulatory molecule by SCC and IFN-gamma by the host promote IL-12 induced regression of SCC.
Angiogenesis Inhibitors↗
Mapping and developmental expression analysis of the WD-repeat gene Preb.
We have isolated from mouse a novel WD-motif-containing gene designated Preb. This gene encodes a predicted protein of 416 amino acids and has significant homology with other members of the WD-motif gene superfamily that play a role in cell fate determination. Preb maps to the proximal end of chromosome 5 in mouse, near the Hmx1 homeobox gene. Preb is detectable in early stage embryos in the peripheral nervous system, developing liver, and surface ectoderm. Later, Preb is expressed in the anterior portion of Rathke's pouch, which gives rise to the anterior pituitary, the organ responsible for the production of prolactin and other hormones. In midgestation embryos, the most extensive expression of Preb is observed in the perichondrium of the craniofacial, axial, and appendicular skeleton. The expression pattern of Preb in murine embryos suggests a potential role in the specification of multiple cell types, in particular, the fetal skeleton.
Amino Acid Sequence↗
Cloning and characterization of human PREB; a gene that maps to a genomic region associated with trisomy 2p syndrome.
We have isolated the human homolog of a novel rodent gene that may be involved in the regulation of pituitary gene transcription. The human PREB gene encodes a predicted protein of 417 amino acids, exhibiting several sequences characteristic of the WD-motif protein family. PREB transcripts were detected in every human fetal and adult tissue examined, although a great variation in levels of expression was observed. PREB was mapped to human Chromosome 2p23, a region of the genome associated with partial trisomy 2p syndrome. Although variable, the common duplication phenotype includes facial abnormalities, skeletal defects, growth and mental retardation, congenital heart and neural tube defects, and abnormalities of the genitalia. We propose that PREB has a role during human development and that abnormal dosage of this transcription factor may be involved in some of the developmental abnormalities observed in patients with partial trisomy 2p.
Amino Acid Sequence↗
Hiding messages in DNA microdots.
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Computing Methodologies↗
Expression cloning and characterization of PREB (prolactin regulatory element binding), a novel WD motif DNA-binding protein with a capacity to regulate prolactin promoter activity.
Previous studies have implied that a transcription factor(s) other than Pit-1 is involved in homeostatic regulation of PRL promoter activity via Pit-1-binding elements. One such element, 1P, was employed to clone from a rat pituitary cDNA expression library a novel 417-amino acid WD protein, designated PREB (PRL regulatory element binding) protein. PREB contains two PQ-rich potential transactivation domains, but no apparent DNA-binding motif, and exhibits sequence-specific binding to site 1P, to a site nonidentical to that for Pit-1. The PREB gene (or a related gene) is conserved, as an apparently single copy, in rat, human, fly, and yeast. A single approximately 1.9-kb PREB transcript accumulates in GH3 rat pituitary cells, to levels similar to Pit-1 mRNA. PREB transcripts were detected in all human tissues examined, but the observation of tissue-specific multiple transcript patterns suggests the possibility of tissue-specific alternative splicing. RT-PCR analysis of human brain tumor RNA samples suggested region-specific expression of PREB transcripts in brain. Western and immunocytochemical analysis implied that PREB accumulates specifically in GH3 cell nuclei. Transient transfection employing PREB-negative C6 rat glial cells showed that PREB is as active as, and additive with, Pit-1 in transactivation of a PRL promoter construct, and that PREB, but not Pit-1, can mediate transcriptional activation by protein kinase A (PKA). Expression in GH3 cells of a GAL4-PREB fusion protein both strongly transactivated a 5XGAL indicator construct and yielded a further stimulation of expression of this construct by coexpressed PKA, implying that PREB can mediate both basal and PKA-stimulated transcriptional responses in pituitary cells. These observations imply that PREB will prove to play a significant transcriptional regulatory role, both in the pituitary and in other organs in which transcripts of its gene are expressed.
Amino Acid Sequence↗
Making DNA add.
Recent studies have demonstrated the feasibility of using DNA-based experiments to compute solutions to combinatorial problems. However, a prerequisite for designing a computer useful in a wide range of applications is the ability to perform mathematical calculations. The development of a DNA-based algorithm for addition is presented. The DNA representation of two nonnegative binary numbers is presented in a form permitting a chain of primer extension reactions to carry out the addition operation. To demonstrate the feasibility of this algorithm, a simple example was executed biochemically.
Algorithms↗
Pituitary adenylate cyclase-activating polypeptide regulates prolactin promoter activity via a protein kinase A-mediated pathway that is independent of the transcriptional pathway employed by thyrotropin-releasing hormone.
The hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide (PACAP), can efficiently increase cAMP levels in pituitary cells and release a number of pituitary hormones, suggesting an important physiological role for this peptide in pituitary function. Exposure of GH3 rat pituitary cells to PACAP results in increases in cellular cAMP levels, PRL promoter activity, and PRL messenger RNA levels. We have employed this system to further characterize PACAP regulation of PRL gene expression. RT-PCR analysis showed that GH3 cells express transcripts for two PACAP receptors, PACAP-R-hop1 and VIP2. As the former can couple PACAP to increases in both cAMP and inositol phosphates, we investigated whether either pathway mediates PACAP action on the PRL promoter. Our observations that TRH, but not PACAP, increases the intracellular Ca2+ concentration in GH3 cell cultures and that the optimal concentrations of TRH and PACAP have additive effects on transient expression of a PRL-CAT construct imply that the inositol trisphosphate-Ca2+ pathway is not significantly involved in PACAP action on the PRL promoter. Four kinase inhibitors exhibited similar profiles of inhibition of the activity on PRL-chloramphenicol acetyltransferase (PRL-CAT) of either the adenylyl cyclase activator forskolin (FSK) or PACAP, suggesting a transcriptional role for protein kinase A (PKA). The observations that coexpression of the dominant PKA inhibitor RAB completely blocked either FSK or PACAP action on PRL-CAT and that these actions of FSK and PACAP were completely nonadditive imply that the cAMP-PKA pathway plays a dominant role in PACAP regulation of PRL gene expression. Coexpression of low levels of KCREB, a cAMP response element (CRE)-binding protein (CREB) dominant inhibitor, partially blocked regulation of PRL-CAT activity by PACAP, but not TRH, implying that PACAP action is mediated at least in part by a CREB family member that can dimerize with CREB. The PRL promoter contains an asymmetric sequence at positions -99/-92 resembling a canonical CRE and termed here the CRE-like element (CLE). Mutation of either the left or right 4 bp of the CLE yielded a strong decrease in the response to either FSK or PACAP, but not to TRH. These data imply that PACAP and TRH employ independent pathways to regulate the PRL promoter, and that PACAP action is exerted virtually entirely via a cAMP/PKA-mediated pathway that is strongly dependent upon an intact CLE sequence and at least partially dependent upon the activity of a CREB-related protein.
Animals↗
Constitutively active G(S) alpha-subunits stimulate Pit-1 promoter activity via a protein kinase A-mediated pathway acting through deoxyribonucleic acid binding sites both for Pit-1 and for adenosine 3',5'-monophosphate response element-binding protein.
Constitutively active mutations of the G protein alpha(S) subunit are detected at a high frequency in human pituitary adenomas that secrete GH or PRL. It seems possible that over-expression of the pituitary cell-specific transcription factor Pit-1/GHF-1 (Pit-1) gene in response to active alpha(S) subunits contributes to the formation of these adenomas. We have examined whether expression in pituitary cells of one of these constitutively active alpha(S) subunits, Q227L-alpha(S), stimulates expression directed by the Pit-1 promoter. Transient expression of Q227L-alpha(S) yielded a strong stimulation of a target Pit-1 promoter-chloramphenicol acetyl transferase (CAT) construct, (-200)Pit-1-CAT. Expression of wild-type alpha(S) or an inactive alpha(S) mutant yielded, respectively, reduced or no stimulation of CAT activity. A dominant inhibitor of protein kinase A (PKA), RAB, blocked almost completely either forskolin (FSK) or Q227L-alpha(S) stimulation of (-200)Pit-1-CAT expression, implying that PKA is required for the action of Q227L-alpha(S) on the Pit-1 promoter. The Pit-1 promoter contains a binding site for Pit-1 and two CREB binding sites. Mutation of the Pit-1 binding site reduced but did not eliminate either FSK or Q227L-alpha(S) stimulation of Pit-1 promoter activity, implying a partial but incomplete requirement for this element for a PKA-mediated response to Q227L-alpha(S). The CREB dominant inhibitor S133A-CREB yielded a partial reduction in either FSK or Q227L-alpha(S) stimulation of (-200)Pit-1-CAT expression, implying that one or both of the Pit-1 promoter adenosine 3'5'-monophosphate response element binding protein (CREB) binding sites is/are also required for a complete PKA-mediated response to Q227L-alpha(S). The observation that S133A-CREB completely blocked the response to FSK or Q227L-alpha(S) of a Pit-1 promoter containing a mutated site PitB1 implies that the binding sites for Pit-1 and CREB account for all of the response elements for FSK or alpha(S) in the Pit-1 promoter.
Animals↗
Constitutively active Gq-alpha stimulates prolactin promoter activity via a pathway involving Raf activity.
We have investigated the ability of a constitutively active Gq-alpha mutant, Q209L-alpha q, to regulate target gene expression. Transient expression in GH3 pituitary cells of a rat proximal prolactin promoter-chloramphenicol acetyltransferase construct (-187)PRL-CAT, was stimulated by co-expression of Q209L alpha q, but not by wild-type alpha q. Q209L-alpha q stimulated expression of constructs driven by promoters for either rat prolactin or growth hormone, but not of a control construct driven by the thymidine kinase promoter. Thus, transcriptional effects of alpha q are specific both for the activated state of this G-alpha subunit and the promoter examined. Since both the prolactin and growth hormone promoters are activated by the pituitary cell-specific transcription factor Pit-1, we examined whether a Pit-1 binding site could direct a response to Q209L-alpha q. Two copies of prolactin promoter Pit-1 binding site 1P conferred upon a heterologous metallothionein promoter a response to Q209L-alpha q, implying an involvement of this site in the transcriptional action of Q209L-alpha q on the prolactin promoter. The phorbol ester activator of protein kinase C, 12-O-tetradecanoylphorbol-13-acetate, stimulated (-187)PRL-CAT activity, but opposed the action of Q209L-alpha q on activity of this PRL-CAT construct. Q209L-alpha q stimulation of (-187)PRL-CAT activity was inhibited by co-expression of a dominant negative Raf mutant, Raf-C4, but not by a point mutant of Raf-C4 with reduced inhibitory properties. These results imply that activated alpha q subunits can stimulate prolactin promoter activity via a pathway that involves a Pit-1 DNA binding site(s), is opposed by protein kinase C, and is mediated by a pathway in which Raf-1 kinase plays a role.
Animals↗
The D2 receptor: blocked transcription in GH3 cells and cellular pathways employed by D2A to regulate prolactin promoter activity.
Although the GH3 line of somatolactotropic rat pituitary cells has proven useful for many regulation studies, the absence of functional D2 receptors on these cells long prevented their use in studies of dopaminergic action. However, it is now possible to employ GH3 cells expressing recombinant D2 receptors for such investigations. We have investigated both the level at which expression of functional D2 receptors in GH3 cells is blocked, and the cellular pathways employed by the major pituitary D2 receptor isoform, D2A, to inhibit prolactin (PRL) gene transcription. In run-off transcription assays with nuclei from either parental GH3 cells or a GH3 cell line stably expressing a D2A expression vector, Pit-1 gene transcription was detectable in either cell line, but only the latter cell line yielded detectable D2 receptor transcription, implying that the block in D2 receptor expression by GH3 cells is transcriptional. Further investigations employed GH3 cells transiently co-transfected with a D2A expression vector plus a rat PRL promoter construct (-1957)PRL-CAT. Pertussis toxin blocked repression by quinpirole, a D2 agonist, of PRL-CAT activity, demonstrating that this action is mediated by a pertussis toxin-sensitive G protein. The observations that neither of two agents expected to raise intracellular Ca2+, Bay K8644 or thyrotropin-releasing hormone, prevented quinpirole repression of PRL-CAT activity, and that the repressive effects on this construct of quinpirole and the Ca2+ channel antagonist were independent, suggested that regulation of intracellular Ca2+ levels does not play a major role in D2A-mediated repression of the PRL promoter.(ABSTRACT TRUNCATED AT 250 WORDS)
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
Nicotine acts directly on pituitary GH3 cells to inhibit prolactin promoter activity.
We have employed the GH3 rat pituitary cell line to investigate whether nicotine can regulate prolactin (PRL) gene expression. Nicotine strongly inhibited (45%) transient expression of a construct containing the first 187 base-pairs of the rat PRL promoter cloned upstream of the chloramphenicol acetyl transferase (CAT) gene. This implies that nicotine acts directly on the GH3 cells to inhibit transcription directed by the PRL promoter. Expression of a control reporter construct containing the CAT gene under the control of the RSV promoter was not affected by exposure of the cells to nicotine, demonstrating that the effect of nicotine is promoter-specific. The inhibition by nicotine of PRL promoter activity was not blocked by hexamethonium, suggesting that this effect of nicotine may be mediated by a novel type of nicotine receptor previously described in frog pituitary cells. Nicotine was also observed to yield a concentration-dependent inhibition of the stimulation by thyrotrophin-releasing hormone (TRH) of PRL promoter activity, implying that nicotine can also interfere with hormonal regulation of the PRL gene. These results suggest that the reduced serum PRL levels that result from smoking may originate in part from decreased transcription of the PRL gene resulting from a direct effect of nicotine on pituitary PRL-secreting cells.
Animals↗
Transcriptional effects in GH3 cells of Gs alpha mutants associated with human pituitary tumors: stimulation of adenosine 3',5'-monophosphate response element-binding protein-mediated transcription and of prolactin and growth hormone promoter activity via protein kinase A.
Somatic mutations of the alpha-subunit of Gs (Gs alpha) have been detected previously at high frequency in human PRL- and/or GH-producing pituitary tumors. To test whether these mutants are responsible for the increased production of these hormones, we used transient cotransfection assays to analyze their genomic effects in GH3 rat pituitary cells. We first show that guanosine triphosphatase (GTPase)-deficient Gs alpha subunits (mutated at amino acid 201 or 227) stimulate transcription from a reporter construct bearing the consensus cAMP response element (CRE; TGACGTCA). Using GAL4-CRE-binding protein fusion constructs, we further show that this stimulatory effects of Gs alpha on the CRE is probably mediated by the transacting factor CRE-binding protein. Then, in experiments using a reporter gene driven by the human promoters for either the PRL (position -250 to 18) or GH (position -500 to 13) genes, we show that these mutant Gs alpha subunits stimulate expression driven by either the PRL or GH promoter. Finally, we show that a dominant inhibitory mutant of cAMP-dependent kinase (protein kinase A) completely blocks the ability of these Gs alpha mutants to stimulate the activity of either the PRL or GH promoter, implying that GTPase-deficient Gs alpha subunits stimulation of the activities of these promoters is mediated entirely via the cAMP/protein kinase A pathway. Taken together, these results imply that activation of this pathway by the GTPase-deficient mutants found in human pituitary tumors stimulates the expression of PRL and GH genes. The transcriptional effects exerted via this pathway may thus provide a basis for the secretory phenotype and endocrine disorders associated with these tumors.
Cyclic AMP Response Element-Binding Protein↗
Expression of constitutively active Gs alpha-subunits in GH3 pituitary cells stimulates prolactin promoter activity.
Although G protein alpha subunits are known to regulate such cellular functions as growth and enzymatic activity, the ability of these proteins to regulate target gene expression has not yet been directly investigated. Transient expression in GH3 pituitary cells of a target rat prolactin promoter-chloramphenicol acetyltransferase construct, (-1957)PRL-CAT, was increased by coexpressed constitutively active alpha s mutant Q227L-alpha s but not by wild-type alpha s. Thus activated alpha s but not basal state alpha s can stimulate prolactin promoter activity. Q227L-alpha s also stimulated expression of construct (-187)PRL-CAT, showing that only the proximal prolactin promoter region is required for a response to activated alpha s. The promoter specificity of the transcriptional influence of activated alpha s was demonstrated by the inability of either Q227L-alpha s or wild-type alpha s to stimulate expression of control target constructs containing either the rat growth hormone promoter or the thymidine kinase promoter. Previous studies have shown that the most proximal prolactin promoter binding site for the pituitary-specific transcription factor pit-1, site 1P, can act as an independent response element for either thyrotropin-releasing hormone or Ca2+. Two copies of site 1P conferred upon a heterologous metallothionein promoter a response to Q227L-alpha s. This implies that site 1P can also serve as an independent response element for alpha s and suggests that pit-1 may be a mediator of the cellular regulation by alpha s of the prolactin promoter.
Animals↗
A constitutively active form of CREB can activate expression of the rat prolactin promoter in non-pituitary cells.
The pituitary cell-specific transcription factor Pit-1 has been show to trans-activate expression of the prolactin (PRL) promoter in non-pituitary cells. However, the cyclic AMP response element (CRE)-binding protein CREB is known to play a major role in cell-specific expression of hepatocyte-specific genes. Since the PRL promoter contains an asymmetrical form of a cyclic AMP response element (termed the CLE), we investigated whether CREB could also induce PRL promoter activity in non-pituitary cells. Transient expression in rat glial C6 cells of a constitutively active CREB-VP16 fusion protein strongly trans-activated expression of a co-transfected rat PRL promoter construct, (-187)PRL-CAT. Analysis by 5'-deletion showed that this response requires PRL promoter sequences between positions -113/-75. CREB-VP16 did not stimulate expression in C6 cells of any of three control promoter-CAT constructs, implying that the strong response of the PRL promoter to activated CREB is both promoter-specific, and is not due to non-specific transcriptional effects of the potent VP16 moiety of CREB-VP16. Surprisingly, mutations in the CLE only slightly reduced activation by CREB-VP16 of construct (-204)PRL-CAT, implying that the major action of CREB-VP16 on the PRL promoter does not involve a direct interaction with the CLE. CREB-VP16 stimulated PRL-CAT activity in C6 cells as strongly as, and synergistically with, Pit-1. These results imply that CREB can strongly and specifically activate expression of the PRL promoter in non-pituitary cells, via a mechanism different from that employed by Pit-1.
Animals↗
A Pit-1 phosphorylation mutant can mediate both basal and induced prolactin and growth hormone promoter activity.
The transcription factor Pit-1 has been shown to be important for both the developmental and homeostatic regulation of expression of the PRL and GH genes in pituitary cells. However, little is known about possible covalent modifications in Pit-1 that might mediate its transactivational properties. Previous studies showing that Pit-1 is a phosphorylation substrate for either protein kinase A or C, or their cellular inducers, led us to investigate whether phosphorylation of Pit-1 is required for its function in either basal or induced cellular activity of either the PRL or GH promoters. The transactivational properties of wild type Pit-1 were compared with those of Pit-1(A3), mutated in the three known phosphorylation sites. At saturating levels of Pit-1 expression vectors, activation of transient basal expression in HeLa cells of constructs (-1957)PRL-CAT or (-244)GH-CAT by RSV-Pit-1(A3) was, respectively, about 50% and 65% as strong as by RSV-Pit-1. Hence, phosphorylation at the sites mutated in Pit-1(A3) is not critically required for basal transactivation of either promoter but may modulate this activity. RSV-Pit-1 and RSV-Pit-1(A3) were equally effective in mediating estrogen receptor stimulation of (-1957)PRL-CAT expression in HeLa cells, thus revealing no phosphorylation requirement for the prerequisite for Pit-1 in estrogen receptor action on the PRL estrogen response element.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Pituitary adenylate cyclase-activating polypeptide stimulates prolactin gene expression in a rat pituitary cell line.
Pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to activate adenylate cyclase and stimulate PRL secretion in dispersed pituitary cells. We have employed the GH3 rat pituitary cell line to investigate whether PACAP can regulate expression of the PRL gene. PACAP increased cellular levels of cAMP in a concentration-dependent fashion (EC50, approximately 6 x 10(-9) M). PACAP also increased PRL mRNA levels in GH3 cells, implying that this peptide stimulates a step in expression of the PRL gene. In addition, PACAP strongly stimulated chloramphenicol acetyltransferase (CAT) activity in GH3 cells transiently transfected with a plasmid containing the first 187 basepairs of the rat PRL promoter cloned up-stream of the CAT gene, implying that PACAP stimulates transcription directed by the PRL promoter. The PACAP stimulation of CAT activity was observed at concentrations as low as 10(-11) M. We examined the action of PACAP on expression of a 5'-deletion series of PRL-CAT constructs. The PACAP response is completely lost when PRL promoter sequences between positions -187 and -113 are removed, implying that neither a previously described sequence resembling a cAMP response element nor the most proximal pit-1-binding site 1P plays a major role in the actions of PACAP on PRL gene transcription. This observation together with the ability of low concentrations of PACAP to stimulate PRL promoter activity without detectably increasing cellular cAMP levels suggest that the action of PACAP on PRL gene transcription might involve a cAMP-independent pathway.
Animals↗
An alternatively spliced Pit-1 isoform altered in its ability to trans-activate.
Although alternative splicing has been shown to give rise to isoforms of a number of transcription factors, such isoforms have not previously been detected for the POU homeodomain protein Pit-1. Screening of a rat pituitary GH3 cell cDNA expression library yielded a clone, termed pCMVPit-1a, encoding a 35.8 kD protein (Pit-1a) containing a 26 amino acid insert in the Pit-1 trans-activation domain. The position of the insert, plus Southern blot analysis, implied that Pit-1a mRNA arises by alternative splicing of the Pit-1 gene transcript. Pit-1a mRNA was detected in GH3 rat pituitary tumor cells at levels about 1/7 that of Pit-1 mRNA. Pit-1a mRNA-specific sequences were also detected in rat and mouse pituitary, and in mouse thyrotropic tumor TtT cells. DNA mobility shift assays showed that Pit-1a binds specifically to Pit-1 binding sites in the proximal prolactin promoter, but produces DNA-protein complexes of markedly different mobilities than Pit-1. In stably transfected CHO cells which accumulated approximately equal levels of either of the two proteins, Pit-1 trans-activated a prolactin promoter-driven CAT construct, while Pit-1a yielded no detectable transactivation, implying a trans-activation ratio for Pit-1a/Pit-1 of less than 0.05. Thus, the insertion of 26 amino acids of similar composition into the activation domain of Pit-1 has at once affected both the mode of binding of this protein and its ability to function as a trans-activator.
Amino Acid Sequence↗