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Purpose: Combining cytotoxic agents with bevacizumab has yielded significant benefits in a number of solid tumors. Combining small-molecule kinase inhibitors of VEGFR with chemotherapy has yet to demonstrate clinical benefit. The dose, schedule and agents used may be critical to the development of this combinatorial therapy.Methods: We performed a phase I trial of sunitinib and gemcitabine in patients with advanced solid tumor malignancies based on strong preclinical rationale.Results: Two different MTDs were determined. The schedule of gemcitabine 800 mg/m(2) on days 1, 8, 15 and sunitinib 25 mg daily was considered to be a MTD. However, omission of day 15 gemcitabine was common, and thus, a second MTD of gemcitabine of 675 mg/m(2) on days 1 and 8 with sunitinib 25 mg daily was determined to be the recommended phase II dose. Grade 4 neutropenia and thrombocytopenia occurred in 33 and 6 %, respectively. Grade 3/4 non-hematological toxicities were uncommon. Four of 33 patients had a partial response. Another 11 patients had stable disease ranging from 3 to 36 months. Thus, the recommended phase II dose of this combination is gemcitabine 675 mg/m(2) on days 1 and 8 on an every 21-day schedule along with sunitinib 25 mg continuous daily.Conclusions: This combination is well-tolerated and has significant clinical activity. [ABSTRACT FROM AUTHOR]

Temozolomide (TMZ) generates DNA adducts that are repaired by direct DNA and base excision repair mechanisms. Methoxyamine (MX, TRC-102) potentiates TMZ activity by binding to apurinic and apyrimidinic (AP) sites after removal of N 3 -methyladenine and N 7 -methylguanine, inhibiting site recognition of AP endonuclease. We conducted a phase I trial to determine the maximum tolerated dose and dose-limiting toxicities (DLTs) of intravenous MX when given with oral TMZ. Patients with advanced solid tumors and progression on standard treatment were enrolled to a standard 3 + 3 dose escalation trial assessing escalating doses of TMZ and MX. Tumor response was assessed per RECIST and adverse events (AEs) by CTCAEv3. Pharmacokinetics (PK) of MX and COMET assays on peripheral blood mononuclear cells were performed. 38 patients were enrolled-median age 59.5 years (38-76), mean number of cycles 2.9 [1-13]. No DLTs were observed. Cycle 1 grade 3 AEs included fatigue, lymphopenia, anemia, INR, leukopenia, neutropenia, allergic reaction, constipation, psychosis and paranoia. Cycle 2-13 grade 4 AEs included thrombocytopenia and confusion. A partial response was seen in 1 patient with a pancreatic neuroendocrine tumor (PNET) and six additional patients, each with different tumor types, demonstrated prolonged stable disease. MX PK was linear with dose and was not affected by concomitant TMZ. TMZ 200 mg/m 2 daily × 5 may be safely administered with MX 150 mg/m 2 intravenously once on day 1 with minimal toxicity. Further studies assessing this drug combination in select tumor types where temozolomide has activity may be warranted.

Introduction: This study aimed to assess the safety and tolerability of the multitargeted tyrosine kinase inhibitor, vandetanib (V), in combination with two chemotherapeutic agents, oxaliplatin (O) and docetaxel (D) in advanced gastroesophageal (GE) cancer., Methods: This was a Phase I study (NCT00732745) with a standard 3+3 dose escalation design. The primary aim was to determine the optimal dose of the combination of vandetanib and OD chemotherapy., Results: Initial treatment for the first cohort consisted of oxaliplatin at 100 mg/m2 on day 1, docetaxel at 35 mg/m2 on days 1 and 8 and vandetanib 100 mg PO daily of 21-day treatment cycles. As dose limiting toxicity (DLT) was reached in 2 out of 3 patients in cohort 1 (one grade 3 and one grade 4 diarrhea with dehydration), 6 patients were treated then at dose level -1 (O 80 mg/m2 on day 1, D 30 mg/m2 on days 1 and 8, V 100 mg PO daily days 1-21) in which no further DLTs were observed. This dose was established as maximum tolerated dose and is the recommended phase 2 dose. 8 out of 9 enrolled patients had adenocarcinoma. At dose level 1, 1 of the 3 patients had a documented partial response and 2 patients had stable disease. At dose level -1, 1 of 6 patients achieved a complete response, 2 of 6 patients had stable disease, and 3 of 6 patients had progressive disease., Conclusions: Vandetanib added to oxaliplatin and docetaxel showed manageable toxicity and limited activity in advanced GE cancer.

The start of the 21st century has produced advances in cancer care that have improved both survival rates and quality of life for many persons diagnosed with cancer. Targeted therapy has given new hope for controlling cancer as a chronic illness. Alone, or in combination with traditional therapies such as surgery, radiation, and/or chemotherapy, this new form of therapy targets malignant cells, halting tumor growth and the potential metastatic spread of disease. Toxicities are limited, but some are serious and may require intensive care. It is imperative for the experienced critical care nurse to have an understanding of these new treatment options and those on the horizon, as these therapies are the future of cancer care. Whereas in previous decades, patients with cancer may not have survived an intensive care admission for treatment complications or advanced disease, many patients now are recovering from life-threatening events, continuing treatment for their disease, and going on to live meaningful, good-quality lives.

Rebeccamycin analog (RA) is an antitumor antibiotic with both topoisomerase I and II inhibiting activity. Topoisomerase inhibitors have demonstrated synergy with platinum agents. We performed a phase I trial of combination RA with oxaliplatin in patients with refractory solid tumors. RA was administered as a 1-hour infusion daily on days 1-5 with oxaliplatin administered on day 5. Cycles were repeated every 21 days. A total of 17 patients were enrolled. The MTD for RA was 80 mg/m(2)/d for five days along with oxaliplatin 130 mg/m(2) on day 5. Myelosuppression was a common occurrence but was mild except in one instance. Dose limiting toxicities included atrial fibrillation and hypophosphatemia. There was evidence of antitumor activity including 3 partial responses in patients with esophageal, gallbladder and hepato-cellular carcinoma; 5 additional patients had stable disease. Thus, the combination of RA and oxaliplatin is both tolerable and has evidence of clinical activity, but given the lack of significant activity for single agent RA across a variety of disease sites, it is unlikely to proceed to phase II development.

Purpose: Cytotoxic and anti-angiogenic drugs are efficacious in malignancies. This trial was undertaken to evaluate the toxicity of a novel regimen combining docetaxel and lenalidomide., Patients and Methods: Patients with advanced solid tumors were eligible. Docetaxel was administered on day 1, and lenalidomide was given on days 1-14 of each 21-day cycle. Since significant myelosuppression occurred, pegfilgrastim was added on day 2. Dose limiting toxicity (DLT) was defined as >or=grade 3 non-hematologic toxicity, grade 4 neutropenia with fever, or grade 4 anemia or thrombocytopenia., Results: Thirty-three patients were enrolled. DLTs included neutropenia, nausea/vomiting, and dyspnea. Of the evaluable patients, 69% had stable disease, and 3% had partial response., Conclusions: This regimen was well tolerated and provided stable disease in the majority of advanced cancer patients. The recommended phase II dosing is docetaxel 75 mg/m(2) on day 1, lenalidomide 25 mg on days 1-14, and pegfilgrastim 6 mg on day 2, given every 3 weeks.

Background: Fosbretabulin is a novel vascular-disrupting agent that has antitumor activity against anaplastic thyroid cancer (ATC) cell lines, xenografts, and demonstrable efficacy in a phase I trial. This phase II study determined the efficacy and safety of fosbretabulin in patients with advanced ATC and whether fosbretabulin altered the natural history of ATC by virtue of doubling the median survival. A secondary aim evaluated the prognostic value of serum soluble intracellular adhesion molecule-1 (sICAM)., Methods: Twenty-six patients received fosbretabulin 45 mg/m(2) as a 10-minute intravenous infusion on days 1, 8, and 15 of a 28-day cycle. sICAM levels were obtained at baseline, over the first two cycles, and end of therapy. Treatment was continued until disease progression., Results: Fosbretabulin was well tolerated; grade 3 toxicity was observed in nine patients (35%), and grade 4 toxicity in one (4%). QTc prolongation delayed treatment in four causing one to stop treatment. Median survival was 4.7 months with 34% and 23% alive at 6 and 12 months, respectively. Median duration of stable disease in seven patients was 12.3 months (range, 4.4-37.9 months). Baseline serum sICAM levels were measured in 24 patients with a median 253.5 ng/mL. There was a significant difference in event-free survival among tertiles of baseline sICAM levels (p < 0.009)., Conclusions: There were no objective responses seen with single-agent fosbretabulin as administered in this trial, and we did not observe a doubling of survival as our primary endpoint. This is among the largest prospective trials ever conducted for ATC. Fosbretabulin has an acceptable safety profile in patients with advanced ATC, and one-third survived more than 6 months. Despite a small sample size, low baseline sICAM levels were predictive of event-free survival. Further prospective validation of sICAM as a therapeutic biomarker and exploring combination regimens with fosbretabulin are warranted.

Purpose: Pre-clinical models have demonstrated the benefit of metronomic schedules of cytotoxic chemotherapy combined with anti-angiogenic compounds. This trial was undertaken to determine the toxicity of a low dose regimen using docetaxel and thalidomide., Patients and Methods: Patients with advanced solid tumors were enrolled. Thalidomide 100mg twice daily was given with escalating doses of docetaxel from 10 to 30 mg/m(2)/week. One cycle consisted of 12 consecutive weeks of therapy. The maximal tolerated dose (MTD) was defined as the dose of thalidomide along with docetaxel that caused < or =grade 1 non-hematologic or < or =grade 2 hematologic toxicity for cycle one., Results: Twenty-six patients were enrolled. Dose-limiting toxicities (DLTs) were bradycardia, fatigue, fever, hyperbilirubinemia, leukopenia, myocardial infarction, and neutropenia. Prolonged freedom from disease progression was observed in 44.4% of the evaluable patients., Conclusions: This anti-angiogenic regimen was well tolerated and demonstrated clinical benefit. The recommended phase II dosing schedule is thalidomide 100 mg twice daily with docetaxel 25 mg/m(2)/week.

N6-methyladenosine (m6A) is present at internal sites in mRNA isolated from all higher eukaryotes, but has not previously been detected in the mRNA of the yeast Saccharomyces cerevisiae. This nucleoside modification occurs only in a sequence- specific context that appears to be conserved across diverse species. The function of this modification is not fully established, but there is some indirect evidence that m6A may play a role in the efficiency of mRNA splicing, transport or translation. The S.cerevisiae gene IME4, which is important for induction of sporulation, is very similar to the human gene MT-A70, which has been shown to be a critical subunit of the human mRNA [N6-adenosine]-methyltransferase. This observation led to the hypothesis that yeast sporulation may be dependent upon methylation of yeast mRNA, mediated by Ime4p. In this study we show that induction of sporulation leads to the appearance of low levels of m6A in yeast mRNA and that this modification requires IME4. Moreover, single amino acid substitutions in the putative catalytic residues of Ime4p lead to severe sporulation defects in a strain whose sporulation ability is completely dependent on this protein. Collectively, these data suggest very strongly that the activation of sporulation by Ime4p is the result of its proposed methyltransferase activity and provide the most direct evidence to date of a physiologic role of m6A in a gene regulatory pathway.

The alternative splicing of the last intron (intron D) of bovine growth hormone (bGH) pre-mRNA requires a down-stream exonic splicing enhancer (FP/ESE). The presence of at least one SR protein has been shown to be essential for FP/ESE function and splicing of intron D in in vitro splicing assays. However, in vitro reconstitution of splicing using individual purified SR proteins may not accurately reflect the true complexity of alternative splicing in an intact nucleus, where multiple SR proteins in varying amounts are likely to be available simultaneously. Here, a panel of recombinant baculovirus-expressed SR proteins was produced and tested for the ability to activate FP/ESE-dependent splicing. Individual recombinant SR proteins differed significantly in their activity in promoting intron D splicing. Among the recombinant SR proteins tested, SRp55 was the most active, SC35 showed very little activity, and ASF/SF2 and 9G8 individually had intermediate activity. At least one SR protein (ASF/SF2) bound to the FP/ESE with characteristics of a cooperative interaction. Most interestingly, low concentrations of ASF/SF2 and 9G8 acted synergistically to activate intron D splicing. This was due in part to synergistic binding to the FP/ESE. Splicing of bGH intron D is inherently complex, and is likely controlled by an interaction of the FP/ESE with several trans-acting protein factors acting both independently and cooperatively. This level of complexity may be required for precise control of alternative splicing by an exon sequence, which simultaneously is constrained to maintain translational integrity of the mature mRNA.

The methylation of internal adenosine residues in eukaryotic mRNA, forming N6-methyladenosine (m6A), is catalyzed by a complex multicomponent enzyme. Previous studies suggested that m6A affects the efficiency of mRNA processing or transport, although the mechanism by which this occurs is not known. As a step toward better understanding the mechanism and function of this ubiquitous posttranscriptional modification, we have shown that HeLa mRNA (N6-adenosine)-methyltransferase requires at least two separate protein factors, MT-A and MT-B, and MT-A contains the AdoMet binding site on a 70-kDa subunit (MT-A70). MT-A70 was purified by conventional chromatography and electrophoresis, and was microsequenced. The peptide sequence was used to design a degenerate oligodeoxynucleotide that in turn was used to isolate the cDNA clone coding for MT-A70 from a HeLa cDNA library. Recombinant MT-A70 was expressed as a fusion protein in bacteria and was used to generate anti-MT-A70 antisera in rabbits. These antisera recognize MT-A70 in HeLa nuclear extracts by western blot and are capable of depleting (N6-adenosine)-methyltransferase activity from HeLa nuclear extract, confirming that MT-A70 is a critical subunit of (N6-adenosine)-methyltransferase. Northern blot analysis reveals that MT-A70 mRNA is present in a wide variety of human tissues and may undergo alternative splicing. MT-A70 cDNA probe hybridizes to a 2.0-kilobase (kb) polyadenylated RNA isolated from HeLa cells, whereas it hybridizes to two predominant RNA species (approximately 2.0 kb and 3.0 kb) using mRNA isolated from six different human tissues. Analysis of the cDNA sequence indicates that it codes for a 580-amino acid protein with a predicted MW = 65 kDa. The predicted protein contains sequences similar to consensus methylation motifs I and II identified in prokaryotic DNA (N6-adenosine)-methyltransferases, suggesting the functional conservation of peptide motifs. MT-A70 also contains a long region of homology to the yeast protein SPO8, which is involved in induction of sporulation by an unknown mechanism.

Human U6 small nuclear RNA (U6 snRNA), an abundant snRNA required for splicing of pre-mRNAs, contains several post-transcriptional modifications including a single m6A (N-6-methyladenosine) at position 43. This A-43 residue is critical for the function of U6 snRNA in splicing of pre-mRNAs. Yeast and plant U6 snRNAs also contain m6A in the corresponding position showing that this modification is evolutionarily conserved. In this study, we show that upon incubation of an unmodified U6 RNA with HeLa cell extract, A-43 residue in human U6 snRNA was rapidly converted to m6A-43. This conversion was detectable as early as 3 min after incubation and was nearly complete in 60 min; no other A residue in U6 snRNA was converted to m6A. Deletion studies showed that the stem-loop structure near the 5' end of U6 snRNA is dispensable for m6A formation; however, the integrity of the 3' stem-loop was necessary for efficient m6A formation. These data show that a short stretch of primary sequence flanking the methylation site is not sufficient for U6 m6A methyltransferase recognition and the enzyme probably recognizes secondary and/or tertiary structural features in U6 snRNA. The enzyme that catalyzes m6A formation in U6 snRNA appears to be distinct from the prolactin mRNA methyltransferase which is also present in HeLa nuclear extracts.

N6-Methyladenosine is found at internal positions of mRNA in higher eukaryotes. This post-transcriptional modification occurs at a frequency of one to three methylation/average mRNA molecule in mammalian cell lines and is sequence-specific. A highly conserved consensus recognition site for the methyltransferase has been determined from both viral and cellular messages, consisting of the sequence Pu(G/A)AC(U/A) (with A being methylated). Despite the ubiquity and the specificity of this modification, little is known about the mechanism of formation of N6-methyladenosine. Utilizing an in vitro methylation system from HeLa cell nuclear extracts, and a substrate RNA derived from the mRNA coding for bovine prolactin, the mRNA N6-adenosine methyltransferase has been characterized and partially purified. Unique among other characterized nucleic acid methyltransferases, the enzyme is composed of three components which are separable under non-denaturing conditions. The molecular masses of the components are 30, 200, and 875 kDa as determined by gel filtration and glycerol gradient sedimentation. The 200-kDa component appears to contain the S-adenosylmethionine-binding site on a 70-kDa subunit. The 875-kDa component has affinity for single-stranded DNA-agarose, suggesting that it may contain the mRNA-binding site. N6-Adenosine methyltransferase is not sensitive to treatment with micrococcal nuclease, nor to immunodepletion using an anti-trimethylguanosine antibody, suggesting that it does not contain an essential RNA component.

The N6-methylation of internal adenosine residues is a common post-transcriptional modification of eukaryotic pre-mRNA sequences. An in vitro methylation system which retains the precise selectivity of in vivo methylation sites has been used to further define the nature of RNA site recognition. In addition to short consensus sequences, other structural features or context effects contribute to the selection of methylation sites in pre-mRNAs. Partial purification of the mRNA N6-adenosine methyltransferase revealed unexpected levels of complexity. The methyltransferase is composed of three separate components with molecular masses of 30, 200 and 875 kDa, respectively. These components are readily separated under non-denaturing conditions and each is required for mRNA methylation activity.

Expression of the glycoprotein hormone alpha-subunit gene occurs in the pituitary of all mammals but in placenta of only primates and horses. In humans, two different elements, termed upstream regulatory element (URE) and cAMP response element (CRE), are required for placenta-specific expression of the alpha-subunit gene. The URE binds a protein unique to placenta whereas the CRE binds a ubiquitous protein. Comparative analysis of the promoter-regulatory region of the alpha-subunit gene from a number of mammals indicates that a functional URE has been retained and suggests the potential for placenta-specific expression. Indirect evidence also indicates that the URE-binding protein has been conserved, even in placenta from mammals that fail to express the alpha-subunit gene. Lack of expression of the alpha-subunit gene in placenta of rodents and cattle can be traced to a single nucleotide change that renders the CRE-like sequence of these genes incapable of binding the protein that confers responsiveness to cAMP. In contrast, although expression of the alpha-subunit gene occurs in horse placenta, the promoter-regulatory region lacks a functional CRE but appears to retain a functional URE. This suggests that either a different accessory element and cognate protein interacts with the horse URE to provide placenta-specific expression or that a completely different set of regulatory elements is required for placenta-specific expression in horses.

cAMP stimulates transcription of the human glycoprotein hormone alpha-subunit gene in choriocarcinoma cells. Combined treatment with phorbol esters potentiates this effect. Tandem cAMP response elements (CREs) in the proximal 5'-flanking sequence mediate the effect of cAMP. In this report, we show that the CREs can also mediate the synergistic effect of phorbol esters. In addition to serving as an inducible cis-acting element, the two CREs act synergistically to increase basal transcription. We now provide direct evidence via equilibrium binding studies that tandem CREs bind their trans-acting factors cooperatively. However, the level of cooperativity is insufficient to explain the high degree of transcriptional synergism, suggesting that another element in the alpha-subunit promoter may be required for complete synergism. In support of this hypothesis, we show that synergism is drastically reduced when the CREs are removed from the context of their native promoter and linked to a heterologous promoter. Thus, amplification of the transcriptional signal mediated by the tandem CREs occurs through at least three distinct mechanisms. First, at the level of signal transduction by convergence of the A- and C-kinase pathways; second, through homotropic interactions of trans-acting factors binding to tandem CREs; and finally through heterotropic interaction of the two CREs with another, as yet, undefined cis-acting element(s) in the human alpha-subunit promoter.

Cyclic AMP responsive elements (CRE) have been identified in several genes, including those encoding the alpha-subunit of glycoprotein hormones and the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK) from the rat. Common to the CRE in these genes is the palindromic sequence T(G/T)ACGTCA. Based upon the strong conservation of this element, we hypothesize that the CRE functions by binding a protein that has been conserved across tissue and species lines. Scatchard analysis of gel mobility shift assays indicate that a nuclear protein in extracts prepared from rat liver and from a human choriocarcinoma cell line binds with high affinity to the cAMP responsive element from either gene (Kd approximately 10(-10) M). In order to identify the critical nucleotides within the CRE from these two genes, a series of oligodeoxynucleotides containing systematic mutations was synthesized and tested for protein binding and transcriptional function. Mutations within the palindromic core of either CRE resulted in a marked loss of binding to the nuclear proteins. Sequences outside the 8-base pair element were less important for nuclear protein binding to the PEPCK CRE and were not important for the alpha-subunit CRE. The relative binding, as determined by gel shift assays, correlated with the ability to confer cAMP responsive transcription to a viral promoter in transfected choriocarcinoma cells. DNase I protection assays suggest that binding of the nuclear factor from rat liver to the PEPCK CRE is more efficient when the core sequence is present in the intact PEPCK promoter regulatory region as compared to the isolated CRE oligodeoxynucleotide. Collectively, these results indicate that the nuclear factors necessary for cAMP induction of transcription of the alpha-subunit and PEPCK genes are conserved between tissues and species. In addition to the conserved features of these cis- and trans-active elements, nonconserved sequences and other elements of the promoter regulatory region influence the affinity of the protein-DNA interaction.

cAMP regulates transcription of the gene encoding the alpha-subunit of human chorionic gonadotropin (hCG) in choriocarcinoma cells (BeWo). To define the sequences required for regulation by cAMP, we inserted fragments from the 5' flanking region of the alpha-subunit gene into a test vector containing the simian virus 40 early promoter (devoid of its enhancer) linked to the bacterial chloramphenicol acetyltransferase (CAT) gene. Results from transient expression assays in BeWo cells indicated that a 1500-base-pair (bp) fragment conferred cAMP responsiveness on the CAT gene regardless of position or orientation of the insert relative to the viral promoter. A subfragment extending from position -169 to position -100 had the same effect on cAMP-induced expression. Furthermore, the entire stimulatory effect could be achieved with an 18-bp synthetic oligodeoxynucleotide corresponding to a direct repeat between positions -146 and -111. In the absence of cAMP, the alpha-subunit 5' flanking sequence also enhanced transcription from the simian virus 40 early promoter. We localized this enhancer activity to the same -169/-100 fragment containing the cAMP response element. The 18-bp element alone, however, had no effect on basal expression. Thus, this short DNA sequence serves as a cAMP response element and also functions independently of other promoter-regulatory elements located in the 5' flanking sequence of the alpha-subunit gene.

The single-copy gene encoding the alpha subunit of glycoprotein hormones is expressed in the pituitaries of all mammals and in the placentas of only primates and horses. We have systematically analyzed the promoter-regulatory elements of the human and bovine alpha-subunit genes to elucidate the molecular mechanisms underlying their divergent patterns of tissue-specific expression. This analysis entailed the use of transient expression assays in a chorionic gonadotropin-secreting human choriocarcinoma cell line, protein-DNA binding assays, and expression of chimeric forms of human or bovine alpha subunit genes in transgenic mice. From the results, we conclude that placental expression of the human alpha-subunit gene requires a functional cyclic AMP response element (CRE) that is present as a tandem repeat in the promoter-regulatory region. In contrast, the promoter-regulatory region of the bovine alpha-subunit gene, as well as of the rat and mouse genes, was found to contain a single CRE homolog that differed from its human counterpart by a single nucleotide. This difference substantially reduced the binding affinity of the bovine CRE homolog for the nuclear protein that bound to the human alpha CRE and thereby rendered the bovine alpha-subunit promoter inactive in human choriocarcinoma cells. However, conversion of the bovine alpha CRE homolog to an authentic alpha CRE restored activity to the bovine alpha-subunit promoter in choriocarcinoma cells. Similarly, a human but not a bovine alpha transgene was expressed in placenta in transgenic mice. Thus, placenta-specific expression of the human alpha-subunit gene may be the consequence of the recent evolution of a functional CRE. Expression of the human alpha transgene in mouse placenta further suggests that evolution of placenta-specific trans-acting factors preceded the appearance of this element. Finally, in contrast to their divergent patterns of placental expression, both the human and bovine alpha-subunit transgenes were expressed in mouse pituitary, indicating differences in the composition of the enhancers required for pituitary- and placenta-specific expression.

The 18-bp direct repeat occurring between positions -146 and -111 in the 5'-flanking region of the human alpha-subunit gene serves two functions: it mediates the transcriptional effect of cAMP and it acts in conjunction with an adjacent cis-acting element (URE) to confer properties of placental-specific expression to the alpha-subunit promoter. Functional activity of the URE and CRE requires binding of a trans-acting factor; each element binds a different factor. Analysis of saturation isotherms provides good evidence that cooperativity is involved in binding of CREB to the 18-bp direct repeat. This cooperativity could account for the synergistic effect of two CRE on both basal and cAMP-stimulated transcription. It remains to be determined whether heterotropic cooperativity is involved in binding of trans-acting factors to the URE and CRE. A major difference between the 5'-flanking region of the human alpha-subunit gene and comparable regions from bovine, rat, and mouse alpha-subunit genes is that the latter contain a single CRE homolog which appears incapable of binding the trans-acting factor that binds to the human alpha CRE. Lack of a functional CRE provides at least one explanation for inactivity of the bovine alpha-subunit promoter in choriocarcinoma cells and probably in bovine placenta as well. Yet, the same bovine promoter-regulatory region that lacks a functional CRE is capable of conferring pituitary-specific expression to the CAT gene in transgenic mice (data not shown). This suggests that the CRE is not required for pituitary-specific expression of the bovine alpha-subunit gene. Instead, another cis-acting element(s) must confer this property to the alpha-subunit promoter. While it is tempting to suggest that bovine, rat, and mouse alpha-subunit genes are not regulated by cAMP because of their inactive CRE homolog, it is also quite possible that other CRE are located further upstream. Accordingly, it will be of interest to obtain additional 5'-flanking sequence and determine whether functional homologs of the human alpha CRE are present in the bovine, rat, and mouse alpha-subunit genes, or whether another class of cis-acting elements provide cAMP-responsiveness.