African-American patients with cancer Talking About Clinical Trials (TACT) with oncologists during consultations: evaluating the efficacy of tailored health messages in a randomised controlled trial-the TACT study protocol.

INTRODUCTION: Low rates of accrual of African-American (AA) patients with cancer to therapeutic clinical trials (CTs) represent a serious and modifiable racial disparity in healthcare that impedes the development of promising cancer therapies. Suboptimal physician-patient consultation communication is a barrier to the accrual of patients with cancer of any race, but communication difficulties are compounded with AA patients. Providing tailored health messages (THM) to AA patients and their physician about CTs has the potential to improve communication, lower barriers to accrual and ameliorate health disparities. OBJECTIVE: (1) Demonstrate the efficacy of THM to increase patient activation as measured by direct observation. (2) Demonstrate the efficacy of THM to improve patient outcomes associated with barriers to AA participation. (3) Explore associations among preconsultation levels of: (A) trust in medical researchers, (B) knowledge and attitudes towards CTs, (C) patient-family member congruence in decision-making, and (D) involvement/information preferences, and group assignment. METHODS AND ANALYSIS: First, using established methods, we will develop THM materials. Second, the efficacy of the intervention is determined in a 2 by 2 factorial randomised controlled trial to test the effectiveness of (1) providing 357 AA patients with cancer with THM with 2 different 'depths' of tailoring and (2) either providing feedback to oncologists about the patients' trial THM or not. The primary analysis compares patient engaged communication in 4 groups preconsultation and postconsultation. ETHICS AND DISSEMINATION: This study was approved by the Virginia Commonwealth University Institutional Review Board. To facilitate use of the THM intervention in diverse settings, we will convene 'user groups' at 3 major US cancer centres. To facilitate dissemination, we will post all materials and the implementation guide in publicly available locations. TRIAL REGISTRATION NUMBER: NCT02356549.

A 3'-transcribed region of the HLA-A2 gene mediates posttranscriptional stimulation by IFN-gamma.

The expression of several MHC class I genes is up-regulated at the transcriptional level by IFN-gamma. Posttranscriptional mechanisms also have been implicated, but not well characterized. To investigate the mechanism of IFN-gamma stimulation of the human MHC class I gene HLA-A2, several human tumor cell lines were transfected with reporter gene constructs driven by the HLA-A2 promoter. We have previously shown that the extended 525-bp HLA-A2 promoter alone, which includes a 5' IFN-stimulated response element consensus sequence, is not sufficient for IFN-gamma response in either K562 or Jurkat cells. In the current study, stable transfection of a genomic HLA-A2 gene construct, containing both 5'- and 3'-flanking sequences, resulted in stimulation of the gene by IFN-gamma. Nuclear run-on assays revealed that, unlike other class I genes, IFN-gamma stimulation of HLA-A mRNA accumulation occurs almost entirely through posttranscriptional mechanisms. RNA stability assays showed that the effect is not mediated by alteration of the half-life of the HLA-A2 mRNA. Formation of the 3' end was unaffected by IFN-gamma treatment. Sequences that mediate the majority of IFN-gamma induction of HLA-A2 mRNA reside in a 127-bp 3'-transcribed region of the gene. This region contains the terminal splice site, the usage of which is not affected by IFN-gamma treatment. These results demonstrate a novel posttranscriptional mechanism of regulation of MHC class I genes by IFN-gamma.

Identification of CCAAT displacement protein (CDP/cut) as a locus-specific repressor of major histocompatibility complex gene expression in human tumor cells.

Human major histocompatibility (MHC) class I antigen expression is important in controlling the metastatic growth of malignant tumors. Locus-specific down-regulation of MHC class I gene expression is frequently observed in human tumors, leading to decreased susceptibility to cytotoxic T-cell-mediated lysis. The mechanism of this down-regulation is incompletely understood. Here, we describe the identification of human CCAAT displacement protein (CDP/cut) as a locus-specific repressor of HLA-B and C gene expression. Transient and stable transfections in HeLa and K562 cells demonstrated the presence of a repressor element 650 base pairs upstream of the first exon of HLA-B7. A specific binding complex with the HLA-B7 and Cw2 repressor elements was demonstrated by EMSA. Formation of the EMSA complex was inhibited specifically with polyclonal antiserum to human CDP/cut, demonstrating that CDP/cut binds the HLA-B7 repressor element. The corresponding region of the HLA-A2 promoter neither repressed HLA-A2 gene expression nor bound CDP/cut. Overexpression of CDP/cut in cell lines deficient in CDP/cut resulted in a nearly 4-fold repression of reporter constructs containing the HLA-B7 repressor element but not the corresponding region of the HLA-A2 promoter. Repression of HLA-B and C gene expression by CDP/cut does not involve displacement of NF-Y, nor is CDP/cut associated with the histone deacetylase HDAC1 when bound to the HLA-B7 repressor element. To our knowledge, these results identify CDP/cut as the first example of a locus-specific repressor of MHC class I gene transcription in human tumor cells.

Modulation of the connexin26 tumor suppressor gene expression through methylation in human mammary epithelial cell lines.

BACKGROUND: Normal mammary epithelial cells express mainly gap junction connexin 26 (Cx26) that is either reduced or absent in breast cancers. Since connexin gene mutations are rare we examined if Cx26 gene repression is related to hypermethylation. MATERIALS AND METHODS: Five breast epithelial cell lines were examined for Cx26 mRNA expression and hypermethylation. Treatment with a DNA methyltransferase inhibitor, 5-Aza-2'-deoxycytidine (5-Aza-CdR), was carried out to determine if Cx26 gene expression could be upregulated. RESULTS: Cx26 expression was easily detectable in an immortalized human mammary epithelial cell line (MCF-10) and markedly diminished (MDA-MB231) or undetectable in (MCF-7, BT-20, T47-D) breast cancer cell lines. Hypermethylation of the Cx26 5' region was observed in MCF-10 and MCF-7 cells. Treatment with 5-Aza-CdR resulted in slight or no induction in Cx26 expression in breast cancer cell lines. CONCLUSIONS: Hypermethylation is unlikely to be a major mechanism for Cx26 gene repression in human mammary cancer cell lines.

Amelioration of retroviral vector silencing in locus control region beta-globin-transgenic mice and transduced F9 embryonic cells.

Retroviral vectors are transcriptionally silenced in hematopoietic stem cells, and this phenomenon must be overcome for effective gene therapy of blood diseases. The murine stem cell virus (MSCV) vector completely silences beta-globin reporter genes regulated by locus control region (LCR) elements 5'HS2 to 5'HS4 in seven of eight transgenic mice. Here, we show that no single known MSCV silencer element is sufficient for complete LCR beta-globin transgene silencing. However, partial silencing of high-copy transgenes is conveyed by the MSCV direct repeat and promoter elements. The CpG methylation pattern of silenced and expressed MSCV promoter transgenes is virtually identical, demonstrating that silencing does not absolutely correlate with methylation status. Combined mutations in all four MSCV silencer elements leads to expression of beta-globin in 6 of 10 transgenic mice. The same mutations incorporated into the HSC1 retrovirus vector direct neo gene expression in 71% of transduced F9 embryonic carcinoma cells. These studies demonstrate that combined mutation of four retroviral silencer elements relieves complete silencing in most transgenic mice and transduced F9 cells and suggests that novel silencer elements remain. Enhanced expression of the HSC1 vector in primitive stem cells is well suited for blood gene therapy applications.

Silencing and activation of embryonic globin gene expression.

An understanding of the mechanisms that control developmental stage-specific transcription of globin genes offers the promise of successful therapeutic activation of fetal or embryonic beta-type genes in beta-thalassemia syndromes. A large body of evidence supports the notion of conservation of such mechanisms across vertebrate species and validates the use of pre-clinical studies of silencing and activation of fetal or embryonic globin genes in animals. Using globin gene transfections into primary avian erythroid cells and cultured murine erythroleukemia cells, we have studied mechanisms involved in stage-specific embryonic beta-type globin gene silencing and activation. These studies show that 1) methylation of the exact CpG nucleotides that are methylated in normal adult erythroid cells in vivo is capable of blocking transcription of a transfected embryonic globin gene promoter via binding of a methyl DNA binding protein in primary erythroid cells. 2) Activation of embryonic beta-type globin gene transcription in adult erythroid cells by short chain fatty acids is mediated through specific DNA sequences both in the promoter and downstream of the promoter.

Intronic and flanking sequences are required to silence enhancement of an embryonic beta-type globin gene.

In the course of studying regulatory elements that affect avian embryonic rho-globin gene expression, the multipotential hematopoietic cell line K562 was transiently transfected with various rho-globin gene constructs containing or lacking an avian erythroid enhancer element. Enhanced levels of rho gene expression were seen from those constructs containing an enhancer element and minimal 5' or 3' flanking rho sequences but were not seen from enhancer-containing constructs that included extensive 5' and 3' flanking sequences. Deletion analysis localized 5' and 3' "enhancer-silencing elements" to -2140 to -2000 and +1865 to +2180 relative to the mRNA cap site. A third element required for enhancer silencing was identified within the second intron of the rho gene. The treatment of K562 cells with hemin, which induces erythroid differentiation, partially alleviated the enhancer-silencing effect. The silencer elements were able to block enhancement from a murine erythroid enhancer, but not from a nonerythroid enhancer. Electrophoretic mobility shift assays demonstrated that the transcription factor YY1 is able to bind both the 5' and 3' enhancer silencer elements; a point mutation of the single overlapping YY1/NF-Y binding site in the 3' element completely abolished the enhancer-silencing effect. These results demonstrate a complex enhancer silencer that requires 5' flanking, intronic, and 3' flanking sequences for a single regulatory effect on a eukaryotic gene.

The human leukocyte antigen A2 interferon-stimulated response element consensus sequence binds a nuclear factor required for constitutive expression.

Both constitutive and interferon-inducible enhancer-like elements have been identified previously in the promoter of human leukocyte antigen (HLA) class I genes. One of these sites is termed the interferon-stimulated response element (ISRE). We have tested the function of an ISRE consensus sequence in the human HLA class I gene HLA-A2 and confirmed previous studies that showed that the HLA-A2 ISRE consensus sequence does not mediate a response to interferons. However, deletion of the ISRE consensus sequence caused a several-fold reduction in the constitutive expression of the HLA-A2 gene in K562 and Jurkat cells. Mobility shift assays performed with the HLA-A2 ISRE revealed the presence of a constitutive binding protein (ISRE/CBP). This protein binds specifically to the HLA-A2 ISRE sequence, and binding is not efficiently competed by the ISRE sequences of the HLA-B7 or ISG54 genes. Substitution of the HLA-B7 or ISG54 ISRE sequences for the HLA-A2 ISRE sequence caused a severalfold reduction in the constitutive expression of the HLA-A2 gene. Mass determinations showed the ISRE/CBP to be 105 kDa, different than any previously characterized ISRE binding proteins. We propose that ISRE/CBP is a novel positive transcriptional regulatory factor for the HLA-A2 gene that may contribute to the differential expression of HLA-A versus HLA-B genes.

Metabolic persistence of fetal hemoglobin.

Hereditary persistence of fetal hemoglobin (HPFH) has typically been ascribed to mutations in the beta-globin gene cluster. Pharmacologic agents, including the short-chain fatty acid butyrate, have been shown to upregulate fetal and embryonic globin gene expression. In this report we investigate the possibility that metabolic derangements characterized by an inability to metabolize another short-chain fatty acid, propionate, could be associated with a persistence of fetal hemoglobin unrelated to alterations in the beta-globin cluster. Embryonic globin gene upregulation in a murine adult erythroid cell culture was shown by RNase protection after induction with three short-chain fatty acids (C2-C5). Chart reviews and measurement of fetal hemoglobin in five patients with abnormalities in propionate (C3) metabolism were undertaken; SSCP/dideoxy fingerprint analysis of the gamma-globin gene promoters was done in three of these five patients. Twelve patients with other metabolic derangements served as controls. Only the four patients with clinically severe abnormalities in propionate metabolism (ages 2 to 11), but without anemia, showed a sustained elevation in fetal hemoglobin (3% to 10%). The level of elevation of fetal hemoglobin in these patients, who lack erythropoietic stress, suggests that propionic acid and/or its metabolites are potent stimulators of fetal hemoglobin expression. Study of this group of patients should allow unique insights into the long-term effects of sustained exposure to elevations of short-chain fatty acid levels.

Butyrate derivatives. New agents for stimulating fetal globin production in the beta-globin disorders.

PURPOSE: Stimulating expression of the normal fetal globin genes is a preferred method of ameliorating sickle cell disease and beta-thalassemia for the majority of patients in North America who do not have appropriate bone marrow donors. PATIENTS AND METHODS: Due to increased survival of red blood cells that contain both hemoglobin S and hemoglobin F, as little as 4-8% fetal globin synthesis in the bone marrow can produce levels of hemoglobin F of approximately 20% in the peripheral circulation. Some success has been achieved in stimulating hemoglobin F using chemotherapeutic agents (such as hydroxyurea and 5-azacytidine) and growth factors (erythropoietin) that alter erythroid growth kinetics. However, there is reluctance to treat children with chemotherapeutic agents because of possible undesirable long-term side effects. RESULTS: Butyric acid and butyrate derivatives are generally safe compounds that stimulate the promoters of individual fetal and embryonic globin genes and thus provide a more specific therapy. An initial trial with the parent compound, given as arginine butyrate, has demonstrated rapid stimulation of fetal globin expression to levels that can ameliorate these conditions. Phase I trials of an oral butyrate derivative with a long plasma half-life have begun. CONCLUSIONS: These agents may provide a new and specific approach for ameliorating the clinical manifestations of sickle cell disease and beta-thalassemia.

Stimulation of MHC class I transcription by interferon-gamma involves a non-A, non-C kinase in addition to protein kinase C.

The signal pathways by which interferon-gamma (IFN-gamma) is able to up-regulate major histocompatibility complex (MHC) class I transcription were studied in two human hematopoietic tumor cell lines, K562 and Ramos. These studies suggest that the IFN-gamma signal is transduced via an H7- and staurosporine-sensitive kinase that is distinct from protein kinase C (PKC) and protein kinase A (PKA) in both cell types. Ramos cells appear to utilize an additional pathway involving double-stranded RNA-dependent protein kinase. PKC and possibly PKA appear to be involved in one or more intersecting pathways by which agonists of these kinases are able to act synergistically with IFN-gamma, but activation of these latter pathways is neither necessary nor sufficient for induction of MHC class I transcription. Modulation of G-protein- and Ca2+-calmodulin-associated pathways and arachidonic acid metabolism had no effect on constitutive or IFN-gamma-stimulated class I transcription. The class I stimulatory factor produced in response to IFN-gamma treatment appears to have a short t1/2. The identity of this factor is unknown, but is likely to be distinct from known mediators of IFN-stimulated transcription. Gene and cell-type specificity in the signal transduction pathways utilized by IFN-gamma implies that such pathways may be useful targets for experimental and therapeutic manipulation.

5'-flanking sequences mediate butyrate stimulation of embryonic globin gene expression in adult erythroid cells.

A stable transfection assay was used to test the mechanism by which embryonic globin gene transcription is stimulated in adult erythroid cells exposed to butyric acid and its analogs. To test the appropriate expression and inducibility of chicken globin genes in murine erythroleukemia (MEL) cells, an adult chicken beta-globin gene construct was stably transfected. The chicken beta-globin gene was found to be coregulated with the endogenous adult mouse alpha-globin gene following induction of erythroid differentiation of the transfected MEL cells by incubation with either 2% dimethyl sulfoxide (DMSO) or 1 mM sodium butyrate (NaB). In contrast, a stably transfected embryonic chicken beta-type globin gene, rho, was downregulated during DMSO-induced MEL cell differentiation. However, incubation with NaB, which induces MEL cell differentiation, or alpha-amino butyrate, which does not induce differentiation of MEL cells, resulted in markedly increased levels of transcription from the stably transfected rho gene. Analysis of histone modification showed that induction of rho gene expression was not correlated with increased bulk histone acetylation. A region of 5'-flanking sequence extending from -569 to -725 bp upstream of the rho gene cap site was found to be required for both downregulation of rho gene expression during DMSO-induced differentiation and upregulation by treatment with NaB or alpha-amino butyrate. These data are support for a novel mechanism by which butyrate compounds can alter cellular gene expression through specific DNA sequences. The results reported here are also evidence that 5'-flanking sequences are involved in the suppression of embryonic globin gene expression in terminally differentiated adult erythroid cells.

Cell-type specificity of interferon-gamma-mediated HLA class I gene transcription in human hematopoietic tumor cells.

Major histocompatibility complex class I gene expression plays a central role in cellular immunity and tumor surveillance. A substantial proportion of spontaneous tumors are class I-deficient and numerous experiments have suggested that alterations in class I expression may alter oncogenicity and, as a result, have potential therapeutic impact. Interferons (IFNs) are able to upregulate class I expression by mechanisms that remain to be elucidated, but which appear to be IFN- and cell-type specific. We have characterized in detail the in vivo class I transcriptional response to IFN-gamma in two human hematopoietic tumor cell lines, the class I-deficient K562 cell line and the class I-positive Ramos cell line. In each, IFN-gamma induces a rapid increase in class I transcription, which is sustained in Ramos cells, but transient in K562 cells. In each, stimulation by IFN-gamma is dependent on ongoing protein synthesis, suggesting the requirement for production of a "primary response" protein. These data suggest that more than one type of IFN-gamma-induced signal is operative in the transcriptional response to IFN-gamma. Cycloheximide alone is also capable of inducing a rapid increase in class I transcription in both cell types, suggesting that constitutive attenuation of class I transcription may be a common phenomenon, and that IFN-gamma may act, in part, by interfering with such attenuation.

BVAC ablative chemotherapy followed by autologous bone marrow transplantation for patients with advanced lymphoma.

Forty-one consecutive patients with lymphoma resistant to conventional combination chemotherapy have been entered into a study in which chemo-ablative therapy and autologous marrow rescue were used with curative intent. The actuarial proportion of 20 patients with Hodgkin's lymphoma remaining alive and free of recurrent disease is 49%, while that for 21 patients with non-Hodgkin's lymphoma is 41%. Our clinical approach to these patients involved a strategy whereby lymphomatous nodes greater than 2 cm in diameter that persisted despite salvage chemotherapy were given boost radiation therapy immediately before chemo-ablation. However, patients with this variable had a significantly lower survival due to septic complications rather than recurrent disease. We conclude that the treatment strategy used in this study with some modification may improve further on the already high probability of long-term disease-free survival experienced by this group of patients.

Allogeneic marrow grafting with partially mismatched, unrelated marrow donors.

Forty patients with advanced hematologic malignancies or severe aplastic anemia received marrow grafts from partially mismatched, unrelated marrow donors. All patients were administered conventional prophylaxis for acute graft-v-host disease (GVHD) consisting of methotrexate and low-dose glucocorticoids. All but two patients who survived at least 30 days showed durable engraftment. Six patients survive 17+ to 36+ months following transplantation. Severe acute GVHD was seen in 47% of the patients; however, no direct correlation between GVHD and the degree of mismatching could be determined. Fatal infections were seen in 29 patients, and in the majority the infection occurred after the granulocyte count had risen to greater than 500 cells/microL. We conclude that the problems encountered in this pilot study can potentially be solved, and that further studies with this type of marrow grafting are warranted.

Negative and positive regulation of human leukocyte antigen class I gene transcription in K562 leukemia cells.

The mechanism of transcriptional activation of human leukocyte antigen class I genes by gamma interferon and 5-azacytidine was studied in K562 human leukemia cells. Nuclear run-on transcription assays with various protein and RNA synthesis inhibitors yield evidence for both stimulation of a positive regulatory factor and inhibition of an mRNA that codes for a labile repressor. A novel mechanism is proposed to explain how 5-azacytidine can activate repressed genes without affecting DNA methylation.

Trans-activation of an upstream early gene promoter of bovine papilloma virus-1 by a product of the viral E2 gene.

The approximately 1000 nucleotide long upstream regulatory region (URR) of bovine papilloma virus-1 (BPV-1) contains a cis element which responds to trans-activation by a diffusible factor encoded in the viral E2 open reading frame (ORF). A series of URR DNA fragments have been linked to two heterologous genes, bacterial chloramphenicol acetyl transferase (cat) or herpes simplex virus-1 thymidine kinase (tk), and tested in transient transfection assays for transcription initiating at the authentic upstream early viral promoter, P89. Transcriptional activity of the P89 promoter was greatly elevated in the presence of the E2 trans-activator gene product. The E2-responsive cis element (E2R) of P89 has been mapped to sequences -277 to -131 nucleotides upstream from the transcription start site (BPV nucleotide 89). The E2R element functioned as a strong transcriptional enhancer in cis with the SV40 early or the tk promoter in the presence, but not in the absence, of the E2 gene product. However, several heterologous promoters which lack sequences related to the E2R element were also trans-activated in transient cotransfections by a function encoded in the E2 ORF of BPV-1, albeit to a much lesser extent. In addition to activation of early viral gene transcription, the E2 regulatory gene(s) may therefore have the potential to alter cellular gene expression.

Preferential in vitro binding of high mobility group proteins 14 and 17 to nucleosomes containing active and DNase I sensitive single-copy genes.

High mobility group (HMG) proteins 14 and 17 bind to mononucleosomes in vitro, but the exact nature of this binding has not been clearly established. A new method was developed to allow direct membrane transfer of DNA from HMG 14/17 bound and unbound nucleosomes, which have been separated by acrylamide gel electrophoresis. Hybridization analysis of membranes obtained by this method revealed that the HMG 14/17 bound nucleosomes of avian erythrocytes and rat hepatic tumor (HTC) cells were enriched, about 2-fold, in actively transcribed genes and also inactive but DNase I sensitive genes. Nucleosomes containing inactive, DNase I resistant genes were bound by HMG 14/17, but not preferentially. Several factors that have been reported to greatly influence the binding of HMG 14/17 to nucleosomes in vitro were tested and shown to not account for the preferential binding to DNase I sensitive chromatin. These factors include nucleosomal linker DNA length, single-stranded DNA nicks, and DNA bulk hypomethylation. An additional factor, histone acetylation, was preferentially associated with the HMG 14/17 bound chromatin fraction of avian erythrocytes, but it was not associated with the HMG 14/17 bound chromatin fraction of metabolically active HTC cells. The latter finding was true for all kinetic forms of histone acetylation.