Human gene marker/therapy clinical protocols.
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Clinical Protocols↗
Biomedical subjects
A B Deisseroth
Publications and source records attributed to A B Deisseroth.
Randomized trial of high-dose chemotherapy and blood cell autografts for high-risk primary breast carcinoma.
BACKGROUND: Uncontrolled studies have reported encouraging outcomes for patients with high-risk primary breast cancer treated with high-dose chemotherapy and autologous hematopoietic stem cell support. We conducted a prospective randomized trial to compare standard-dose chemotherapy with the same therapy followed by high-dose chemotherapy. PATIENTS AND METHODS: Patients with 10 or more positive axillary lymph nodes after primary breast surgery or patients with four or more positive lymph nodes after four cycles of primary (neoadjuvant) chemotherapy were eligible. All patients were to receive eight cycles of 5-fluorouracil, doxorubicin (Adriamycin), and cyclophosphamide (FAC). Patients were stratified by stage and randomly assigned to receive two cycles of high-dose cyclophosphamide, etoposide, and cisplatin with autologous hematopoietic stem cell support or no additional chemotherapy. Tamoxifen was planned for postmenopausal patients with estrogen receptor-positive tumors and chest wall radiotherapy was planned for all. All P values are from two-sided tests. RESULTS: Seventy-eight patients (48 after primary surgery and 30 after primary chemotherapy) were registered. Thirty-nine patients were randomly assigned to FAC and 39 to FAC followed by high-dose chemotherapy. After a median follow-up of 6.5 years, there have been 41 relapses. In intention-to-treat analyses, estimated 3-year relapse-free survival rates were 62% and 48% for FAC and FAC/high-dose chemotherapy, respectively (P =.35), and 3-year survival rates were 77% and 58%, respectively (P =.23). Overall, there was greater and more frequent morbidity associated with high-dose chemotherapy than with FAC; there was one septic death associated with high-dose chemotherapy. CONCLUSIONS: No relapse-free or overall survival advantage was associated with the use of high-dose chemotherapy, and morbidity was increased with its use. Thus, high-dose chemotherapy is not indicated outside a clinical trial.
Adult↗
Human gene marker/therapy clinical protocols.
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Clinical Protocols↗
The presence of the Rb c-box peptide in the cytoplasm inhibits p210bcr-abl transforming function.
In order to test if the carboxyl terminal polypeptide of the Retinoblastoma (Rb) tumor suppressor protein, could be used to suppress the growth factor-independent growth phenotype of p210bcr-abl positive myeloid cells, we introduced a truncated form of the 3' end of the Rb cDNA encoding its last 173 amino acid residues (Rb C-box) which localize into the cytoplasm where the p210bcr-abl transforming protein is found, into myeloid cells (32D) which depends on the p210bcr-abl protein for IL3 growth factor-independent growth (32D-p210). The expression of the plasmid vectors carrying the Rb C-box cDNAs was shown to inhibit the abl tyrosine specific protein kinase activity of the p210(bcr-abl) oncoprotein and to suppress the IL3-independent growth phenotype of the 32D-p210 cells. The Rb C-box polypeptides did not suppress the growth of the untransfected 32D parental cell line in methylcellulose in the presence of IL3-conditioned medium. These results suggest that the cytoplasmic localization of the p210(bcr-abl) allows it to escape the effect of intranuclear proteins such as Rb which negatively regulate the p145(c-abl) kinase.
Animals↗
Thiotepa, busulfan and cyclophosphamide as a preparative regimen for allogeneic transplantation for advanced chronic myelogenous leukemia.
Thirty-six adults with chronic myelogenous leukemia (CML) in second or greater chronic phase, accelerated phase, or blast crisis underwent marrow or blood stem cell transplantation from an HLA-matched sibling using high-dose thiotepa, busulfan and cyclophosphamide (TBC) as the preparative regimen. All evaluable patients engrafted and had complete donor chimerism. One patient failed to clear meningeal leukemia, and one patient had one of 30 metaphases positive for the Philadelphia chromosome at 2 months post transplant. The remainder of the patients studied had eradication of CML documented by cytogenetics and/or Southern blot for BCR gene rearrangement, and 13 of 15 patients studied became negative for the BCR gene rearrangement by polymerase chain reaction. Three-year relapse rate is 42% (95% CI, 19-64%). The relapse rate was significantly lower for patients transplanted without blast crisis (9% vs 100%, P < 0.001). Eight (22%, 95% CI, 10-39%) patients had severe or fatal veno-occlusive disease (VOD). Elevated liver enzymes within 1 month prior to transplantation and transplantation using marrow were significantly associated with the occurrence of VOD. Three-year survival is 28% (95% CI, 13-43%). Survival was significantly higher for patients transplanted without blast crisis (45% vs 0%, P = 0.01). TBC is an effective preparative regimen for CML in accelerated phase but not refractory blast crisis, and it should be used with caution in patients with prior hepatopathy who have an increased risk of severe VOD.
Adult↗
L-selectin expression enhances clonogenesis of CD34+ cord blood progenitors.
L-selectin, a surface adhesion glycoprotein expressed on leukocytes, has a well-established role in mediating inflammation and lymphocyte recirculation. Recent evidence suggests that L-selectin may also influence hematopoiesis. We observed that a greater proportion of CD34+ cells express L-selectin in cord blood compared with adult bone marrow, and we hypothesized that L-selectin expression is associated with enhanced clonogenic properties. To test this, we compared CD34+/L-selectin+ cells with CD34+/L-selectin- cells in hematopoietic clonogenic assays. From CD34+/L-selectin+ cell cultures, we observed a 3-fold increase of d 12-14 colony-forming unit-granulocyte/macrophage and multipotent progenitor cells, and a 5-fold enhancement of primitive d 21 high proliferative potential colony-forming cells compared with the progeny of CD34+/L-selectin- cells. We conclude that CD34+ cord blood cells expressing L-selectin are enriched in their clonogenic activity compared with cell fractions lacking L-selectin expression.
Adult↗
Hematopoietic retroviral gene marking in patients with follicular non-Hodgkin's lymphoma.
We conducted a double retroviral vector (RV) gene marking trial to test for the possible contribution to relapse of follicular non-Hodgkin's lymphoma (FNHL) cells present in bone marrow (BM) and peripheral blood (PB) grafts used for hematopoietic reconstitution of patients undergoing myelaoblative chemotherapy and autologous transplant. CD34 positive selection using the CellPro Ceprate CD34 column was performed on PB mononuclear cells obtained after cyclophosphamide/G-CSF mobilization. CD34 positive cells were exposed for 4-6 hours to the LNL6 or G1 Na RV in the absence of growth factors or stromal monolayers. One week later, BM mononuclear cells were similarly processed. Patients then received total body irradiation (TBI), cyclophosphamide, and etoposide followed by infusion of both PB and BM CD34 positive cells. Semiquantitative Southern blot analysis of DNA t(14;18) amplification products showed approximately a three log reduction in t(14;18) positive cells after CD34 positive selection. The first patient showed evidence of engraftment with RV positive BM and PB cells for 9 months. He relapsed one year after transplant. At relapse, one year after transplant, he had lost evidence of RV positive cells in ficolled mononuclear BM and PB cells as well as in CD19 positive cells. The second and third patients showed evidence of engraftment with RV positive cells up to 9 and 6 months post BMT respectively. The second and third patients are still in clinical remission. Our results demonstrate engraftment of RV transduced hematopoietic cells in the PB and BM for up to 9 months.
Adult↗
Presence of P210bcrabl is associated with decreased expression of a beta chemokine C10 gene in a P210bcrabl-positive myeloid leukemia cell line.
BACKGROUND: Chronic myelogenous leukemia (CML) is thought to start with the acquisition of the t(9;22) chromosomal translocation that codes for the P210bcrabl tyrosine-specific protein kinase. The CML cells exhibit anchorage-independent cell growth and genetic instability. After the initial phase, the cells acquire the phenotype of growth factor-independent growth. After the chronic phase, the disease evolves into the accelerated and blastic phases through the process of sequential random mutation. MATERIALS AND METHODS: To identify some of the genetic changes that contribute to the phenotype of blastic and accelerated phase cells, we used differential display PCR to compare levels of cDNA reverse transcripts of mRNA in 32Dc13 cells and 32Dc13 cells that were stably transfected with a bcrabl cDNA plasmid in a constitutively expressed transcription unit. These cells were designated 32Dc13P210bcrabl. For these studies, we used the 32D myeloid leukemia cell line, which depends on IL-3 for growth. RESULTS: Following introduction of the bcr-abl cDNA through transfection, the cell line became growth factor independent, mimicking the change in phenotype that occurs during the later phases of CML. These differential display screening assays detected altered levels of transcripts for 28 genes. Of interest to the biology of growth factor-independent growth in the bcrabl-positive 32D cells was the fact that the C10 beta chemokine gene was expressed at higher levels in the 32Dc13 cells than in the 32Dc13P210bcrabl cells. CONCLUSIONS: These studies show that a C10 beta chemokine gene was expressed at different levels with or without P210bcrabl.
Base Sequence↗
Clinical trials involving multidrug resistance transcription units in retroviral vectors.
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ATP Binding Cassette Transporter, Subfamily B, Mem↗
Peptide containing the BCR oligomerization domain (AA 1-160) reverses the transformed phenotype of p210bcr-abl positive 32D myeloid leukemia cells.
We first showed that the introduction of a bcr-abl transcription unit into the 32D murine myeloid cell line (P210bcrabl32D) converts this cell line from an IL3 dependent cell line to an IL3 growth independent cell line. We next cloned a fragment of the bcr-abl cDNA, which codes for the bcr oligomerization domain and neighboring regions. To test for a transformation inhibitory effect of this oligomerization inhibitory peptide transcription unit on the p210bcr-abl mediated IL3 independent growth of the P210bcrabl32D cell line, we transiently co-electroporated into the growth factor dependent 32D cells, mixtures of plasmids which contained varying ratios of the plasmid expression vectors for the bcr oligomerization inhibitory peptide along with a smaller amount of the plasmid expression vector for the full length p210bcr-abl. (The P210bcr-abl protein converts the 32D from a growth factor dependent into a growth factor independent cell line.) We then showed that the oligomerization domain containing fragment from the bcr and bcr-abl proteins, can be used to inhibit the IL3 independent growth of p210bcr-abl positive 32D cells. These studies may be of eventual interest for those investigators whose goal is to design molecular therapeutic approaches to CML based on the use of peptidomimetic chemical functionalities, which mimic the structure and the inhibitory binding properties of the oligomerization domain containing fragment so as to inhibit the transforming function of the P210bcr-abl oncoprotein.
Animals↗
Cytosine deaminase adenoviral vector and 5-fluorocytosine selectively reduce breast cancer cells 1 million-fold when they contaminate hematopoietic cells: a potential purging method for autologous transplantation.
Ad.CMV-CD is a replication incompetent adenoviral vector carrying a cytomegalovirus (CMV)-driven transcription unit of the cytosine deaminase (CD) gene. The CD transcription unit in this vector catalyzes the deamination of the nontoxic pro-drug, 5-fluorocytosine (5-FC), thus converting it to the cytotoxic drug 5-fluorouracil (5-FU). This adenoviral vector prodrug activation system has been proposed for use in selectively sensitizing breast cancer cells, which may contaminate collections of autologous stem cells products from breast cancer patients, to the toxic effects of 5-FC, without damaging the reconstitutive capability of the normal hematopoietic cells. This system could conceivably kill even the nondividing breast cancer cells, because the levels of 5-FU generated by this system are 10 to 30 times that associated with systemic administration of 5-FU. The incorporation of 5-FU into mRNA at these high levels is sufficient to disrupt mRNA processing and protein synthesis so that even nondividing cells die of protein starvation. To test if the CD adenoviral vector sensitizes breast cancer cells to 5-FC, we exposed primary explants of normal human mammary epithelial cells (HMECs) and the established breast cancer cell (BCC) lines MCF-7 and MDA-MB-453 to the Ad.CMV-CD for 90 minutes. This produced a 100-fold sensitization of these epithelial cells to the effects of 48 hours of exposure to 5-FC. We next tested the selectivity of this system for BCC. When peripheral blood mononuclear cells (PBMCs), collected from cancer patients during the recovery phase from conventional dose chemotherapy-induced myelosuppression, were exposed to the Ad.CMV-CD for 90 minutes in serum-free conditions, little or no detectable conversion of 5-FC into 5-FU was seen even after 48 hours of exposure to high doses of 5-FC. In contrast, 70% of 5-FC was converted into the cytotoxic agent 5-FU when MCF-7 breast cancer cells (BCCs) were exposed to the same Ad.CMV-CD vector followed by 5-FC for 48 hours. All of the BCC lines tested were shown to be sensitive to infection by adenoviral vectors when exposed to a recombinant adenoviral vector containing the reporter gene betagalactosidase (Ad.CMV-betagal). In contrast, less than 1% of the CD34-selected cells and their more immature subsets, such as the CD34+CD38- or CD34(+)CD33- subpopulations, were positive for infection by the Ad.CMV-betagal vector, as judged by fluorescence-activated cell sorting (FACS) analysis, when exposed to the adenoviral vector under conditions that did not commit the early hematopoietic precursor cells to maturation. When artificial mixtures of hematopoietic cells and BCCs were exposed for 90 minutes to the Ad.CMV-CD vector and to 5-FC for 10 days or more, a greater than 1 million fold reduction in the number of BCCs, as measured by colony-limiting dilution assays, was observed. To test if the conditions were damaging for the hematopoietic reconstituting cells, marrow cells collected from 5-FU-treated male donor mice were incubated with the cytosine deaminase adenoviral vector and then exposed to 5-FC either for 4 days in vitro before transplantation or for 14 days immediately after transplantation in vivo. There was no significant decrease in the reconstituting capability of the male marrow cells, as measured by their persistence in female irradiated recipients for up to 6 months after transplantation. These observations suggest that adenovirus-mediated gene transfer of the Escherichia coli cytosine deaminase gene followed by exposure to the nontoxic pro-drug 5-FC may be a potential strategy to selectively reduce the level of contaminating BCCs in collections of hematopoietic cells used for autografts in breast cancer patients.
Adenoviridae↗
Loss of imprinting in disease progression in chronic myelogenous leukemia.
The pathophysiologic role of the Philadelphia chromosome translocation in chronic myelogenous leukemia (CML) has been known for nearly 20 years. However, the most significant morbidity and mortality in CML are caused by progression to blast crisis, about which comparatively little is known at the molecular level. Genomic imprinting is a chromosomal modification leading to parental-origin-specific gene expression in somatic cells. Recently, we and others have described loss of imprinting (LOI) of the insulin-like growth factor-II gene (IGF2), leading to biallelic rather than monoallelic expression in a wide variety of solid tumors. We have now examined the imprinting status of IGF2 in samples from CML patients in stable phase, accelerated phase, and blast crisis. Five of six stable-phase patients showed normal imprinting, but LOI was found in all six cases of advanced disease (three accelerated phase, three blast crisis), which was statistically highly significant (P < .01). Thus, LOI represents a novel type of genetic alteration in CML that appears to be specifically associated with disease progression.
Blast Crisis↗
Rescue from apoptosis in early (CD34-selected) versus late (non-CD34-selected) human hematopoietic cells by very late antigen 4- and vascular cell adhesion molecule (VCAM) 1-dependent adhesion to bone marrow stromal cells.
Monoclonal antibodies to very late antigen 4 (VLA-4) recognize the alpha4beta1 integrin receptor. This monoclonal antibody blocks the adhesion between early hematopoietic progenitor cells (CD34-selected cells) and stromal cells when added to cultures of these cells. Addition of the VLA-4 monoclonal antibody to cultures of stromal cells and CD34-selected cells was shown to induce apoptosis of CD34-selected cells in these CD34-selected cell/stromal cell cocultures, as measured by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling method. In contrast to these experiments with early hematopoietic progenitor cells (CD34+), the level of adhesion between more differentiated cells (unfractionated hematopoietic cells) and stromal cells was not significantly altered by addition of the anti-VLA-4 monoclonal antibody. Similarly, the level of apoptosis of unfractionated hematopoietic cells was not significantly increased by the addition of anti-VLA-4 monoclonal antibody to cultures of the latter cells with stromal cells. The binding of the unfractionated cells is less than that of the CD34-selected cells. Given that there is no difference between the alpha4beta1 integrin expression level of the early and late myeloid cells, there may be a difference in the functional state of the integrin between the early and late myeloid cells. We also show that CD34+-selected precursor cells proliferate at a higher rate when these cells are plated on recombinant vascular cell adhesion molecule 1 molecules. These data indicate that the alpha4beta1 integrin receptor (VLA-4) plays a central role in the apoptosis rescue function that results from the anchorage-dependent growth of the CD34-selected early hematopoietic cells on stromal cells. The data suggest that these apoptosis rescue pathways have less significance as the cells mature and become anchorage independent in their growth. These data should assist in the design of systems for the ex vivo proliferation and transduction of early hematopoietic cells for genetic therapy.
Antibodies, Monoclonal↗
Ratio of bcl-xshort to bcl-xlong is different in good- and poor-prognosis subsets of acute myeloid leukemia.
BACKGROUND: Acute myeloid leukemia (AML) is a heterogeneous collection of leukemic disorders ranging from chemotherapy-sensitive subsets [inversion 16 and t(8;21)], which often can be cured with cytosine arabinoside alone, to the most resistant subsets, which can survive even supralethal levels of combination alkylator chemotherapy (cytogenetic subsets monosomy 5 and monosomy 7). MATERIALS AND METHODS: To analyze the expression of BCL-2 family genes, which are expressed in these subsets of AML, we used PCR sequence amplification reactions that are dependent on oligonucleotide primers representing the BH1 and BH2 homology domains to generate the unique regions between BH1 and BH2. These primers are conserved among all members of the BCL-2 gene family and are separated by a 150 nucleotide region sequence between the BH1 and BH2 domains. The PCR products unique to each BCL-2 family member were cloned directionally into sequencing vectors. The identity of the insert of each clone was determined by slotblots of the DNA amplified from individual colonies and by hybridization with radioactive probes specific to the bcl-2, bcl-x, or bax genes. RESULTS: We found that bcl-2 is the predominant member expressed in AML samples with a poor prognosis (-5, -7), whereas the transcripts of bcl-x are higher than those of bcl-2 in the AML samples with a good prognosis [inv16, t(8;21)]. No significant difference in bax expression was detected between AML subsets of good and bad prognosis. The ratio of bcl-xlong, which inhibits apoptosis, to bcl-xshort, which promotes apoptosis, was determined by amplification with a pair of primers specific to bcl-x followed by separation of the PCR product on agarose gels. Bcl-xlong and bcl-xshort appeared as bands of different molecular mass on a molecular weight gel and were visualized by ethidium bromide staining or Southern blot analysis with a bcl-x-specific probe. CONCLUSIONS: We found that the ratio of bcl-x long to bcl-x short was higher in the AML patients with a poor prognosis. These experiments showed that the levels of BCL-2 family members in the leukemia cells of good- and poor-prognosis subsets are different. In addition, novel members of the BCL-2 family were isolated from the cells of AML patients of either prognosis.
Acute Disease↗
Low and maximally phosphorylated levels of the retinoblastoma protein confer poor prognosis in newly diagnosed acute myelogenous leukemia: a prospective study.
A prior retrospective study suggested that the level of retinoblastoma protein (RB) expression was prognostic for survival in acute myelogenous leukemia (AML). Individuals with no/low RB protein expression were considered to have loss of RB function, and those with maximally phosphorylated (maxphos) RB were also felt to have nonfunctional RB. To confirm this, we prospectively investigated whether the level of RB expression was prognostic in AML in a larger cohort of patients. RB level was measured by Western blot and immunohistochemical analysis on peripheral blood samples from 210 newly diagnosed AML patients. Patients were divided into three groups based on the level of RB protein expression (i.e., no or low, elevated, and maxphos) or into two groups on the basis of presumed RB function, altered function (AF-RB, low and maxphos RB), versus normal function (NF-RB, elevated RB). By combined results of Western blot and immunohistochemical analysis, 20%, 65%, and 15% of patients had low, elevated, and maxphos RB, respectively. Most patients with acute promyelocytic leukemia (APL) with a French-American-British classification of M3 were in the low RB group, likely reflecting a lower proliferative rate of promyelocytes. Analysis was performed with and without these APL patients. The median survival was significantly shorter for both patients with low RB expression (48 weeks, P = 0.05, including APL patients; 34 weeks, corrected P = 0.008, with APL patients excluded) and maxphos RB expression (51 weeks, P = 0.007) compared to those with elevated RB expression (122 weeks including and 98 weeks excluding APL patients). Differences were greatest among patients with nonfavorable prognosis cytogenetics (median survival, 34 weeks versus 85 weeks; corrected P = 0.001 for AF-RB versus NF-RB). Remission duration was also significantly shorter for non-APL patients with AF-RB versus NF-RB (median survival, 36 weeks versus not reached; corrected P = 0.02). In multivariate analyses, including cytogenetics, performance status, age, antecedent hematological disorder, and RB status, with and without APL patients included, no/low and maxphos-RB protein expression were independent predictors for poorer survival. This prospective study confirms that the level of expression of RB is a strong prognostic factor in AML, with an inferior survival experience being associated with no/low RB and maxphos RB expression. Therefore, therapeutic decisions based on the level of RB expression may be indicated, and protocols to incorporate this are currently under development.
Adolescent↗
Human gene marker/therapy clinical protocols.
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Genetic Markers↗
Expression of epidermal growth factor receptor and human papillomavirus E6/E7 proteins in cervical carcinoma cells.
BACKGROUND: Epidermal growth factor receptor (EGF-R) proteins are highly expressed in many tumors, including those of the cervix. We have observed previously that the introduction of a transcription unit containing an antisense sequence for the E6/E7 genes of human papillomavirus (HPV) 18, along with a transcription unit containing a sense complementary DNA sequence for the wild-type retinoblastoma (Rb) gene, decreased the growth of human cervical carcinoma HeLa cells (HPV 18 positive) both in vitro and in vivo. To clarify the regulatory mechanisms by which this reduction in cell proliferation occurred, we studied the expression of EGF-R proteins in these cells. METHODS: Western blot and northern blot techniques were used to measure EGF-R expression, and a pulse-chase immunoprecipitation assay was used to measure the stability of EGF-R protein in HeLa cells and HeLa cells that had been transfected with the antisense E6/E7 or sense Rb sequences. Cell proliferation was measured by use of a tetrazolium-based colorimetric assay for numbers of viable cells. RESULTS: The introduction of sense Rb or antisense E6/E7 transcription units or a combination of these two transcription units into HeLa cells dramatically decreased the level of EGF-R proteins in these cells; EGF-R levels were not affected at the transcriptional level but at the post-transcriptional level. Addition of the anti-EGF-R-specific monoclonal antibody 225mAb to HeLa cells caused 53% (95% confidence interval = 44%-62%) growth inhibition. CONCLUSIONS: These results suggest that HeLa cervical carcinoma cells are dependent on EGF-R for proliferation and that changes in functional levels of the E6/E7 HPV proteins and endogenous Rb proteins may alter the growth rate of cervical cancer cell lines by reducing the stability of EGF-R at the post-transcriptional level.
Blotting, Northern↗
Use of the cosmid adenoviral vector cloning system for the in vitro construction of recombinant adenoviral vectors.
The large size of the adenoviral genome unfortunately precludes there being many unique, useful restriction sites available for in vitro manipulation. Two methods have been developed for the construction of recombinant adenoviral vectors to date: in vivo homologous recombination or direct ligation in vitro. The efficiency of either the direct ligation method or the homologous recombination method is low because of the large size of the recombinant adenoviral vectors. To circumvent these problems, we have chosen to use the cosmid vector system to facilitate the assembly of recombinant adenoviral vectors. In this paper, we demonstrate for the first time that recombinant adenoviral vectors can be efficiently constructed in vitro by the cosmid vector system. With this method, it is possible to amplify the recombinant adenoviral vector DNA sufficiently to transfect 293 cells. The cosmid adenoviral vector cloning method for in vitro construction of the full-length recombinant adenoviral vectors represented here is simple and efficient and should facilitate the development of recombinant adenoviral vectors for human gene therapy.
Adenoviridae↗