Biomedical subjects

A B Deisseroth

Publications and source records attributed to A B Deisseroth.

At least 73 records · Page 4Linked to original sources

PubMed1995

Transduction of MDR1 into human and mouse haemopoietic progenitor cells: use of rhodamine (Rh123) to determine transduction frequency and in vivo selection.

The MDR1 gene product P-glycoprotein (P-gp) extrudes several anticancer drugs including taxol and fluorescent dyes such as rhodamine (Rh123). Modulation of the level of P-gp expression has the potential of overcoming multidrug resistance. One possible approach is the retroviral transfer of the human MDR1 gene into murine and human bone marrow (BM) progenitor cells. The rationale for this approach is increased chemoprotection, which allows chemotherapy of a greater level of intensity to be delivered. In this study, flow cytometric measurement of Rh123 extrusion was used to test P-gp function in human and mouse haemopoietic progenitor cells, which had been transduced with a virus containing the human MDR1 transcription unit. Human CD34+ selected cells were analysed immediately following transduction. In two successive experiments MDR1 cDNA transduction resulted in a 7% and 11% increase of P-gp expressing Rh123 dull cells. To monitor transduction efficiency over time as well as the possibility of in vivo selection of drug-resistant BM cells in mice treated with increasing numbers of taxol cycles, the assay was also successfully applied to peripheral blood lymphocytes of mice transplanted with MDR1 transduced BM cells, demonstrating increased Rh123 efflux in transduced cells. Analysis of another fluorescence assay using fluorescein di-beta galactopyranoside as a substrate for beta-galactosidase in cells transduced with a MDR1: beta-gal activity. We conclude that the Rh123 efflux assay is a sensitive method to monitor P-gp function in MDR1 cDNA transduced cells, and may be used to enrich transduced cells via flow cytometric cell sorting for Rh123 dull cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Wild-type human p53 and a temperature-sensitive mutant induce Fas/APO-1 expression.

Fas/APO-1 is a cell surface protein known to trigger apoptosis upon specific antibody engagement. Because wild-type p53 can activate transcription as well as induce apoptosis, we queried whether p53 might upregulate Fas/APO-1. To explore this possibility, we examined human p53-null (H358 non-small-cell lung adenocarcinoma and K562 erythroleukemia) and wild-type p53-containing (H460 non-small-cell lung adenocarcinoma) cell lines. When H358 or H460 cells were transduced with a replication-deficient adenovirus expression construct containing the human wild-type p53 gene but not with vector alone, a marked upregulation (approximately a three-to fourfold increase) of cell surface Fas/APO-1 was observed by flow cytometry. Similarly, K562, cells stably transfected with a plasmid vector containing the temperature-sensitive human p53 mutant Ala-143 demonstrated a four- to sixfold upregulation of Fas/APO-1 by flow-cytometric analysis at the permissive temperature of 32.5 degrees C. Temperature-sensitive upregulation of Fas/APO-1 in K562 Ala-143 cells was verified by immunoprecipitation and demonstrated to result from enhanced mRNA production by nuclear run-on and Northern (RNA) analyses. Stably transfected K562 cells expressing temperature-insensitive, transcriptionally inactive p53 mutants (His-175, Trp-248, His-273, or Gly-281) failed to upregulate Fas/APO-1 at either 32.5 degrees or 37.5 degrees C. The temperature-sensitive transcription of Fas/APO-1 occurred in the presence of cycloheximide, indicating that de novo protein synthesis was not required and suggested a direct involvement of p53. Collectively, these observations argue that Fas/APO-1 is a target gene for transcriptional activation by p53.

Adenocarcinoma↗

PubMed1995

Collection of peripheral-blood diploid cells from chronic myelogenous leukemia patients early in the recovery phase from myelosuppression induced by intensive-dose chemotherapy.

PURPOSE: To evaluate whether intensive chemotherapy followed by peripheral stem-cell (PSC) collections during early hematopoietic recovery results in a higher percentage of diploid cell collections in patients with Philadelphia chromosome (Ph)-positive chronic myelogenous leukemia (CML). PATIENTS AND METHODS: Fifty-five adults with Ph-positive CML received intensive chemotherapy with daunorubicin and high-dose cytarabine (ara-C) (DAUNO-HDAC; 26 patients) or fludarabine, high-dose ara-C, and mitoxantrone (FAM; 29 patients). Collections of the peripheral mononuclear cells were initiated when the WBC count was > or = 0.8 x 10(3)/microL. Simultaneous peripheral and marrow samples were subjected to cytogenetic studies. RESULTS: Thirty-eight of 55 patients (69%) were able to undergo the PSC collections. The rate of collection was higher in chronic phase (26 of 30 patients; 87%) than in accelerated (11 of 17; 65%) and blastic phases (1 of 8; 12%). Among the 30 patients in chronic phase, cytogenetic analyses of PSC showed cytogenetic responses (Ph-positive < 95%) in 60%, which were major (Ph < 35%) in 43% and complete (Ph = 0%) in 27%. Seven of 19 patients with simultaneous studies (37%; 23% of total) had a significantly lower percentage of Ph-positive cells in the peripheral collection compared with the marrow collection; one had the reverse phenomenon (5%; 3% of total). Cytogenetic responses were modest in both peripheral and marrow collections in CML accelerated and blastic phases. Myelosuppression-associated complications were frequent, resulting in febrile episodes in 76% of patients. CONCLUSION: PSC collection during early hematopoietic recovery from intensive chemotherapy allowed the collection of diploid-rich stem cells, mostly in chronic-phase CML. The approach could be used for in vivo purging before autologous stem-cell transplantation (ASCT).

Adolescent↗

PubMed1995

Growth-factor stimulation reveals two mechanisms of retinoblastoma gene inactivation in human myelogenous leukemia cells.

Mutation or deletion of the retinoblastoma tumor suppressor gene (Rb) or abnormal Rb protein expression is found in many types of human solid tumors. Low or absent levels of Rb protein are usually found in the leukemic cells of patients with acute myelogenous leukemia (AML) who have an extremely poor prognosis. The absence of Rb protein in these AML cells could result from defects in the Rb gene or from abnormal cell cycle regulation that affects Rb expression. To test these possibilities and to examine whether a low level of Rb protein in AML cells could be up-regulated, we studied the effect that growth factors interleukin 3 (IL3) and granulocyte-macrophage colony stimulating factor (GM-CSF) had on the levels of Rb protein and Rb phosphorylation in AML cells from patients with low Rb or no Rb protein expression. We observed three responses to growth factor-stimulation in leukemic cells taken from patients with AML: (1) some AML cell samples entered a proliferative phase, and Rb protein levels increased with the appearance of normally phosphorylated forms of Rb protein and positive nuclear staining for Rb protein; (2) some AML cell samples became more proliferative, but the levels of Rb protein remained low or absent; and (3) some AML cell samples showed no response. These results indicate that at least two different mechanisms may be responsible for the lack of Rb protein in the leukemic cells of some patients with AML.

Gene Expression Regulation, Leukemic↗

PubMed1995

Resistance to taxol chemotherapy produced in mouse marrow cells by safety-modified retroviruses containing a human MDR-1 transcription unit.

We used an animal model system to transplant lethally-irradiated mice with one million marrow cells which had been: (1) collected from 5-fluorouracil (5-FU) treated mice; and (2) transduced with retroviruses containing a multiple drug resistance-1 (MDR-1) gene transcription unit. Following recovery from the transplant, we exposed these mice to doses of taxol ranging from 7 mg/kg to 30 mg/kg, which corresponds to doses of 68 to 268 mg/m2 in man. The median white blood cell count by 5 days after taxol (expressed as the percentage of the white blood cell count before taxol) was 83% (range 46-100%) in 11 courses of taxol in mice transplanted once with MDR-1 transduced marrow immediately after transplant, whereas the median white blood cell count by 5 days after taxol in mice not transplanted with MDR-1 marrow was 41% in nine courses of taxol (range 11-66%). This difference is statistically different at the P < 0.001 level (Wilcoxon test). One million marrow cells from the MDR-1 transplanted mice were harvested and serially transplanted through five additional cohorts of mice, and tested with taxol after each cohort. The white blood cell count (expressed as the percentage of pre-taxol white blood cell count) after each cohort ranged from 94-146% in the 29 mice transplanted with the transduced MDR-1 marrow, which had been through more than one transplant. This is statistically different from the median white blood cell count recovery after taxol in mice which have no human MDR-1 modified marrow (P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Suppression of tumorigenesis by transcription units expressing the antisense E6 and E7 messenger RNA (mRNA) for the transforming proteins of the human papilloma virus and the sense mRNA for the retinoblastoma gene in cervical carcinoma cells.

Human cervical carcinoma cell lines that harbor human papilloma virus (HPV) have been reported to express HPV E6 and E7 proteins at least in the beginning stages if not at all stages of the disease. The HPV E6 and E7 proteins bind to and inactivate the products of the p53 and retinoblastoma (Rb) tumor suppressor genes, which thereby allow the cervical carcinoma cells to circumvent the action of these tumor suppressor genes. We observed that the introduction of the antisense HPV 18 E6 and E7 sequences, as well as a sense cDNA for the human wild-type Rb gene into a human cervical carcinoma cell line (HeLa), which is positive for the HPV 18 provirus, decreased the in vitro and in vivo growth rate of the transfected cells if both antisense transcripts for the HPV 18 E6 and E7 and sense transcripts for human Rb were expressed. In addition, overexpression of a complementary DNA (cDNA) for the Rb messenger RNA was sufficient to slow the proliferation of HeLa cells, and the level of Rb cDNA expression was correlated with the degree to which the rate of growth of the tumor was slowed. The results of our experiments show that the presence of HPV E6 and E7 proteins and the resultant inactivation of Rb in cervical carcinoma cells contributes to the neoplastic phenotype even in highly evolved cervical carcinoma cell lines such as HeLa, which have been derived from a cervical carcinoma patient at an advanced stage of the disease process. These data suggest that the HPV proteins play a role not only at the beginning of cervical cancer, but also at advanced stages of this disease. These experiments may lead to genetic approaches to the control of this disease that involve antisense sequences that downregulate the E6 and E7 genes or lead to expression of the Rb gene.

Animals↗

PubMed1995

Prevention of graft-versus-host disease with anti-CD5 ricin A chain immunotoxin after CD3-depleted HLA-nonidentical marrow transplantation in pediatric leukemia patients.

To determine if partial T cell depletion and intensive post-transplant immunosuppression is effective for the prevention of graft-versus-host disease (GVHD) in pediatric recipients of HLA-non-identical marrow transplants, 10 children with leukemia received high-dose thiotepa, cyclophosphamide and total body irradiation followed by transplantation of CD3-depleted marrow from matched unrelated or one-antigen mismatched related adult donors. To maximize the number of stem cells infused, a large volume (1-1.51) of marrow was harvested from the donors. After immunopurging, the marrow infused contained a median of 3.7 x 10(6) CD34+ cells/kg, 1.4 x 10(6) CD3+ cells/kg, and 1.6 x 10(6) CD5+ cells/kg as assessed by flow cytometry. Cyclosporine, methylprednisolone and anti-CD4 ricin A chain immunotoxin (XZ-CD5) were used for prevention of GVHD post-transplant. All patients achieved an ANC > 0.5 x 10(9)/l. No patient developed capillary leak syndrome or renal failure from XZ-CD5. Five developed grade 2-4 acute GVHD, and all responded to treatment with steroids. Five of nine evaluable patients developed chronic GVHD. Two patients relapsed, but the most common cause of death was infection with or without chronic GVHD. Four patients survive 10+ to 27+ months post-transplant. XZ-CD5 is well-tolerated in T cell-depleted marrow transplant recipients. However, partial T cell depletion and intensive post-transplant immunosuppression did not prevent moderate acute GVHD or chronic GVHD. This may have been due to the high number of T cells infused with the marrow.

Adolescent↗

PubMed1995

PML/RARalpha, a fusion protein in acute promyelocytic leukemia, prevents growth factor withdrawal-induced apoptosis in TF-1 cells.

A unique mRNA produced by the t(15;17) (q22-24;q11-21) translocation in the leukemic cells of acute promyelocytic leukemia patients encodes a chimeric protein, PML/RARalpha, which is formed by the fusion of the retinoic acid receptor alpha (RARalpha) and the promyelocytic locus gene (PML). This translocation is often the only visible karyotypic aberration present which is detected in almost 100% of acute promyelocytic leukemia patients. As an initial step to study the role of PML/RARalpha in leukemogenesis, we attempted to express the fusion protein in hematopoietic cells through retrovirus-mediated gene transfer of the retroviral vector, pGPRCHT, which contains the PML/RARalpha cDNA. Transduction of the PML/RARalpha cDNA fragment used in this vector, which extends from the position 31 bp to the position 2638 bp in a transcription unit driven by the Moloney murine sarcoma virus LTR, was found to abrogate the growth factor dependence of TF-1 cells. In addition, introduction of PML/RARalpha into TF-1 cells can protect these cells from the apoptosis usually induced in TF-1 cells by growth factor withdrawal, as measured by three assays for apoptosis: morphology, DNA ladder formation, and end labeling of nicked DNA with fluorescent-conjugated nucleotide precursors followed by a fluorescence-activated cell sorting assay. These data suggest that the PML/RARalpha fusion protein may inhibit programmed cell death in myeloid cells.

Apoptosis↗

PubMed1995

High levels of constitutive WAF1/Cip1 protein are associated with chemoresistance in acute myelogenous leukemia.

The WAF1/Cip1 gene product is an important regulator at the G1 checkpoint in the cell cycle. WAF1/Cip1 expression can be activated through p53-dependent and p53-independent pathways. The WAF1/Cip1 protein binds to cyclin-dependent kinase complexes and inhibits the kinase activity that is required for cell cycle progression. In this preliminary study, we analyzed with Western blot assays the steady-state levels of the WAF1/Cip1 protein in the leukemia cells of 100 untreated acute myelogenous leukemia (AML) patients. Normal bone marrow cells from six donors were used as a control. The results of these analyses showed that the levels of the WAF1/Cip1 protein were very low in normal marrow cells and in the leukemia cells of 83 AML patients. High levels of WAF1/Cip1 were detected in 17 patients; these patients with high WAF1/Cip1 levels were significantly less likely to achieve complete remission (41% versus 69%, P = 0.03) and were four times as likely to be resistant to therapy (47% versus 12%, P = 0.003) as patients with very low levels of WAF1/Cip1. Median survival was 38 weeks for patients having very low expression levels versus 11 weeks for patients having high expression levels (P = 0.04). The WAF1/Cip1 level was an independent predictor for response but not survival in a stepwise multivariate regression analysis. Southern blotting analyses did not detect deletion of the WAF1/Cip1 gene in the 12 negative WAF1/Cip1 AML samples tested. Also, the level of WAF1/Cip1 protein expression was not correlated with overexpression of cyclin D1, cyclin E, proliferating cell nuclear antigen, cyclin-dependent kinase 4, or p53 in the leukemia cells. However, the levels of cyclin D1, cyclin E, and cyclin-dependent kinase 4 were elevated in most of the AML samples compared with that in normal marrow. We hypothesize that high-level constitutively expressed WAF1/Cip1 in tumor cells may result in an indolent state that is refractory to chemotherapy drugs. We conclude that the WAF1/Cip1 expression level may be an important prognostic factor for response to therapy and survival in AML patients.

Adult↗

PubMed1994

Hyperphosphorylation of p53 induced by okadaic acid attenuates its transcriptional activation function.

The tumor suppressor and transcriptional factor p53 is a phosphorylated protein. Its phosphorylation states are regulated by several protein kinases and phosphatases. In this study, the wild-type p53 was transfected and expressed in chronic myelogenous leukemia K-562 cells. Incubation of the transfected cells with okadaic acid, an inhibitor of serine phosphatases 2A and 1, induced hyperphosphorylation of p53 protein. The treatment also increased the steady state level of p53 protein in the cells. However, the hyperphosphorylated p53 protein was less active in promoting transcription mediated by two p53-binding DNA elements, the ribosomal gene cluster and the p53 consensus DNA-binding sequence. Nevertheless, the decreased transcription activation was not due to decreased binding of p53 to these elements, as analyzed by mobility shift DNA-binding assays. In addition, the treatment did not induce a conformational change in p53, as assayed by two conformation-specific anti-p53 monoclonal antibodies, PAb240 and PAb1620. These results suggest that the phosphorylation induced by okadaic acid may selectively modulate the transcription activation function of p53. Consequently, phosphorylation may represent a mechanism of p53 inactivation in tumor cells that harbor the wild-type p53.

Base Sequence↗

PubMed1994

Levels of retinoblastoma protein expression in newly diagnosed acute myelogenous leukemia.

The relationship between the level of retinoblastoma protein (RB) expression and the survival of 113 newly diagnosed acute myelogenous leukemia (AML) patients was studied. Western blotting was used to determine the level of RB protein present in peripheral blood leukemia cells and results were confirmed in 26 patients by immunohistochemistry. The leukemic cells from 22/113 AML patients (19%) contained RB protein at levels that were equal to or less than the level of RB observed in the mononuclear cell fraction of peripheral blood from normal individuals (Low RB). Levels of RB greater than that of normal blood (Elevated RB) were seen in 91 patients (81%). The median survival of patients with low RB was significantly shorter than that seen in patients with elevated RB, 12 weeks versus 40 weeks (P = .02). Remission induction frequency was 36% in low RB patients compared with 68% in AML patients with elevated RB (P = .01). Multivariate analysis showed that low RB protein level was an independent prognostic factor predictive or poor survival after allowing for other known prognostic factors. These data suggest that a low level of the RB protein at the time of diagnosis is associated with shortened survival in AML patients because of inferior response to conventional therapy. Monitoring of the RB level could identify a subgroup of AML patients with an extremely poor prognosis when treated with chemotherapy alone, who would be eligible for alternative therapeutic strategies.

Adolescent↗

PubMed1994

BCR-ABL protein expression in peripheral blood cells of chronic myelogenous leukemia patients undergoing therapy.

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder associated with the Philadelphia chromosome (Ph1) in more than 95% of these patients. The Ph1 and the resulting BCR-ABL fused genes are markers for this type of leukemia. In CML, the product of the fused BCR-ABL gene is typically a protein of approximately 2,000 amino acids termed P210 BCR-ABL. We have developed an assay for the BCR-ABL protein involving Western blotting of circulating white blood cells (WBC) with an anti-ABL monoclonal antibody that can detect P210 BCR-ABL and P145 ABL in peripheral blood cells from chronic phase Ph1-positive leukemia patients. This assay was used to analyze the BCR-ABL protein content of circulating WBC from CML patients before and after various treatments. In parallel to changes in percentages of Ph1-positive blood cells as determined by cytogenetic analyses of bone marrow samples, BCR-ABL protein expression in blood cells decreased or increased as patients entered remission or underwent relapse. Of interest, six Ph1-negative CML patients were BCR-ABL protein-positive. All except one had a rearrangement in the major breakpoint cluster region and that patient expressed P185 BCR-ABL and not P210. Our results indicate that the BCR-ABL Western blotting assay has clinical applications for both diagnosis and prospective evaluation of Ph1-positive and Ph1-negative CML patients.

Fusion Proteins, bcr-abl↗

PubMed1994

A temperature-sensitive mutant of human p53.

We discovered that the human 'hot-spot' p53 mutant 143Ala is temperature sensitive for the binding of two DNA elements and for mediating the transcription of a downstream luciferase reporter gene. At 32.5 degrees C, 143Ala possesses strong DNA binding ability. In addition, its transcriptional activities are stronger than those of wild-type p53. At 37.5 degrees C, however, both the mutant's DNA binding and transcriptional activation functions are greatly reduced or abolished. The activity differences correlate with the presence of two conformational states of p53, which are recognized by monoclonal antibodies: at 32.5 degrees C, 143Ala is recognized by PAb1620, which is specific for the wild-type conformation, whereas at 37.5 degrees C, 143Ala is almost undetectable by PAb1620-mediated immunoprecipitation. Although 143Ala's binding to p53 DNA recognition elements and its activation of reporter gene transcription at 32.5 degrees C is markedly higher than that of the wild-type p53, 143Ala inhibited proliferation less robustly than wild-type p53 and it did not increase inhibition of ras-induced focus formation. These results indicate a partial correlation between the events involved in transcriptional activation and events involved in the proliferation suppression of p53. 143Ala therefore will be of use in dissecting the relationship between the structure of p53 and its different cellular functions.

Animals↗

PubMed1994

Genetic marking shows that Ph+ cells present in autologous transplants of chronic myelogenous leukemia (CML) contribute to relapse after autologous bone marrow in CML.

Relapse after autologous bone marrow transplantation for chronic myelogenous leukemia (CML) can be due either to the persistence of leukemia cells in systemic tissues following preparative therapy, or due to the persistence of leukemia cells in the autologous marrow used to restore marrow function after intensive therapy. To help distinguish between these two possible causes of relapse, we used safety-modified retroviruses, which contain the bacterial resistance gene NEO, to mark autologous marrow cells that had been collected from patients early in the phase of hematopoietic recovery after in vivo chemotherapy. The cells were then subjected to ex vivo CD34 selection following collection and 30% of the bone marrow were exposed to a safety-modified virus. This marrow was infused after delivery of systemic therapy, which consisted of total body irradiation (1,020 cGy), cyclophosphamide (120 mg/kg), and VP-16 (750 mg/m2). RT PCR assays specific for the bacterial NEO mRNA, which was coded for by the virus, and the bcr-abl mRNA showed that in two evaluable CML patients transplanted with marked cells, sufficient numbers of leukemia cells remained in the infused marrow to contribute to systemic relapse. In addition, both normal and leukemic cells positive for the retroviral transgenome persisted in the systemic circulation of the patients for at least 280 days posttransplant showing that the infused marrow was responsible for the return of hematopoiesis following the preparative therapy. This observation shows that it is possible to use a replication-incompetent safety-modified retrovirus in order to introduce DNA sequences into the hematopoietic cells of patients undergoing autologous bone marrow transplantation. Moreover, this data suggested that additional fractionation procedures will be necessary to reduce the probability of relapse after bone marrow transplantation in at least the advanced stages of the disease in CML patients undergoing autologous bone marrow transplantation procedures.

Antigens, CD↗

PubMed1994

Analysis of interphase cells for the Philadelphia translocation using painting probe made by inter-Alu-polymerase chain reaction from a radiation hybrid.

Fluorescence in situ hybridization (FISH) probe for the identification of the Philadelphia (Ph) translocation [t(9;22) (q34;q11)] in chronic myelogenous leukemia cells was developed by inter-Alu-polymerase chain reaction of DNA from an interspecific somatic cell hybrid containing approximately 5 Mb of human DNA covering the ABL gene region on human chromosome 9q34. This probe was large enough to be effective in identifying the genomic domains yet small enough to resolve them in more than 90% of bone marrow interphase cells. Combination of the probe with a cosmid contig probe for the BCR region of chromosome 22 in two-color FISH reduced the frequency of false-positive identification of the Ph chromosome to less than 1%. The procedure allows detection of as few as 1% Ph+ cells independent of the cycling status or BCR/ABL expression level of cells, and the quantitation of non-Ph chromosome-containing interphase nuclei in the marrow of patients judged 100% Ph+ by standard cytogenetics.

Humans↗

PubMed1994

Detection of fusion transcripts generated by the inversion 16 chromosome in acute myelogenous leukemia.

Pericentric inversion of chromosome 16 [inv(16)(p13q22)] and the related t(16;16)(p13;q22) are seen in a subset of acute myelogenous leukemia (AML) phenotypically and prognostically differing from other cases. We have recently shown that inv(16) results in fusion of CBFB/PEBP2B, a gene encoded at 16q22 to MYH11, a smooth muscle myosin heavy chain gene encoded at 16p13. Chimeric transcripts consisting of upstream CBFB fused to downstream MYH11 coding sequences result from this fusion. In this study we have examined a series of 37 of these cases using reverse transcriptase-polymerase chain reaction (RT-PCR) to detect expression of a hybrid CBFB/MYH11 transcript. Chimeric cDNAs were detected in all but 1 of 37 leukemias with typical inv(16) or t(16;16). Such chimeric products were not seen in a case with inv(16)(p13q24) (ie, a variant q arm breakpoint) or any of 10 cases of AML without these chromosomal changes. Four different chimeric transcripts were found, representing differing fusion points within MYH11 spliced to position 495 of CBFB. Primer sets are described for efficient amplification of these different cDNA forms. Amplification of cDNA showed that all but 17 codons of the CBFB coding sequence are included in the abnormal transcripts. RT-PCR was shown to be highly sensitive and potentially useful for detection of leukemic cells during morphologic remission.

Base Sequence↗

PubMed1994

Human interferon regulatory factor 2 gene. Intron-exon organization and functional analysis of 5'-flanking region.

Interferon regulatory factor 2 (IRF-2) is a transcriptional regulatory protein that terminates interferon beta expression initiated by interferon regulatory factor 1. In this study, we isolated the genomic DNA for human IRF-2 gene, determined the intron-exon structure of the human IRF-2 gene, mapped the major transcription initiation site, identified a number of potential regulatory elements in the 5'-flanking region, and localized the IRF-2 gene on human chromosome 4. The IRF-2 promoter region contains a CpG island, with several GC boxes, a putative NF-kappa B-binding site, and a CAAT box, but no TATA box. When the promoter region was linked with a heterologous reporter gene, we found that the promoter region is inducible by both interferons (interferon-alpha and -gamma) and interferon regulatory factor 1. The region which induced these inductions was identified as being confined to 40 nucleotides 5' to the major transcriptional initiation site by testing a series of clones with truncated promoter of IRF-2. This region contains elements which are shared with the transcriptional enhancers of other genes including interferon regulatory factor 1, interferon beta, and interferon-inducible genes. These data suggest that interferon regulatory factor 1 not only triggers the activation of the interferon signal transduction pathway, but also may play a role in limiting the duration of this response by activating the transcription of IRF-2.

Amino Acid Sequence↗

PubMed1994

Retinoblastoma protein expression is frequently altered in chronic lymphocytic leukemia.

The detection of abnormalities at the retinoblastoma (RB) locus by cytogenetics, Southern blot, and fluorescence in situ hybridization studies suggests that the RB gene has a role in chronic lymphocytic leukemia (CLL). To further study this role, we determined the level of RB protein present in the mononuclear cell fraction derived from peripheral blood or bone marrow samples from 74 patients with CLL, by Western blotting. Compared to similarly prepared samples from the peripheral blood of normal individuals, the level of RB in CLL cells was less than normal in 42% of patients, equal to normal in 22% of patients, and in excess of normal in 36% of patients. Regardless of whether the source of the sample was blood or marrow or if the patients were untreated or previously treated, similar rates of low, normal, and elevated RB levels were observed. RB protein in the CLL patient samples was never phosphorylated. RB levels showed no correlation with the lymphocyte doubling time or with proliferating cell nuclear antigen levels. Low RB levels could arise from genetic alterations of the RB gene or altered regulation of expression. To determine which was occurring, we stimulated the cells from 27 CLL patients in culture with either phytohemagglutinin or pokeweed mitogen in an attempt to induce RB expression and phosphorylation. Among patients with low levels of RB, expression was induced in 46% (6 of 13), and phosphorylation of RB was seen in 31% (4 of 13). Increased expression of phosphorylated RB was induced in 80% (4 of 5) of patients with normal levels of RB and in 78% (7 of 9) of patients with high levels of RB. This study demonstrates that absent RB expression occurs commonly in patients with CLL. Intrinsic abnormalities of the RB gene may be present in those patients with low levels of RB that could not be stimulated by mitogens, while regulatory abnormalities located in trans to the RB gene may occur in the other half. Given the importance that RB levels play in other cancers, the prognostic implication of low RB levels should be studied in CLL.

Blotting, Western↗