[Show abstract][Hide abstract] ABSTRACT: Aldehyde dehydrogenase (ALDH) activity has been implicated in multiple biological and biochemical pathways and has been used to identify potential cancer stem cells. Our main hypothesis is that ALDH activity may be a lung cancer stem cell marker. Using flow cytometry, we sorted cells with bright (ALDH(br)) and dim (ALDH(lo)) ALDH activity found in H522 lung cancer cell line. We used in vitro proliferation and colony assays as well as a xenograft animal model to test our hypothesis. Cytogenetic analysis demonstrated that the ALDH(br) cells are indeed a different clone, but when left in normal culture conditions will give rise to ALDH(lo) cells. Furthermore, the ALDH(br) cells grow slower, have low clonal efficiency, and give rise to morphologically distinct colonies. The ability to form primary xenografts in NOD/SCID mice by ALDH(br) and ALDH(lo) cells was tested by injecting single cell suspension under the skin in each flank of same animal. Tumor size was calculated weekly. ALDH1A1 and ALDH3A1 immunohistochemistry (IHC) was performed on excised tumors. These tumors were also used to re-establish cell suspension, measure ALDH activity, and re-injection for secondary and tertiary transplants. The results indicate that both cell types can form tumors but the ones from ALDH(br) cells grew much slower in primary recipient mice. Histologically, there was no significant difference in the expression of ALDH in primary tumors originating from ALDH(br) or ALDH(lo) cells. Secondary and tertiary xenografts originating from ALDH(br) grew faster and bigger than those formed by ALDH(lo) cells. In conclusion, ALDH(br) cells may have some of the traditional features of stem cells in terms of being mostly dormant and slow to divide, but require support of other cells (ALDH(lo)) to sustain tumor growth. These observations and the known role of ALDH in drug resistance may have significant therapeutic implications in the treatment of lung cancer.
[Show abstract][Hide abstract] ABSTRACT: Methylmalonic aciduria (MMA) is an autosomal recessive disease with symptoms that include ketoacidosis, lethargy, recurrent vomiting, dehydration, respiratory distress, muscular hypotonia and death due to methylmalonic acid levels that are up to 1000-fold greater than normal. CblB MMA, a subset of the mutations leading to MMA, is caused by a deficiency in the enzyme cob(I)alamin adenosyltransferase (ATR). No animal model currently exists for this disease. ATR functions within the mitochondria matrix in the final conversion of cobalamin into coenzyme B(12), adenosylcobalamin (AdoCbl). AdoCbl is a required coenzyme for the mitochondrial enzyme methylmalonyl-CoA mutase (MCM).
The human ATR cDNA was cloned into a recombinant adeno-associated virus (rAAV) vector and packaged into AAV 2 or 8 capsids and delivered by portal vein injection to C57/Bl6 mice at a dose of 1 x 10(10) and 1 x 10(11) particles. Eight weeks post-injection RNA, genomic DNA and protein were then extracted and analyzed.
Using primer pairs specific to the cytomegalovirus (CMV) enhancer/chicken beta-actin (CBAT) promoter within the rAAV vectors, genome copy numbers were found to be 0.03, 2.03 and 0.10 per cell in liver for the rAAV8 low dose, rAAV8 high dose and rAAV2 high dose, respectively. Western blotting performed on mitochondrial protein extracts demonstrated protein levels were comparable to control levels in the rAAV8 low dose and rAAV2 high dose animals and 3- to 5-fold higher than control levels were observed in high dose animals. Immunostaining demonstrated enhanced transduction efficiency of hepatocytes to over 40% in the rAAV8 high dose animals, compared to 9% and 5% transduction in rAAV2 high dose and rAAV8 low dose animals, respectively.
These data demonstrate the feasibility of efficient ATR gene transfer to the liver as a prelude to future gene therapy experiments.
The Journal of Gene Medicine 06/2007; 9(6):462-9. DOI:10.1002/jgm.1040 · 2.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Published cytogenetic data for extant cetacean species remain incomplete. In a review of the literature, we found karyotypic information for 6 of the 13 tentatively recognized species of the suborder Mysticeti (baleen whales). Among those yet to be described is the critically endangered North Atlantic right whale (Eubalaena glacialis). Herein, we describe and propose a first-generation G-banded karyotype and ideogram for this species (2n = 42), obtained from peripheral blood chromosome preparations from a stranded male calf. This information may prove useful for future genetic mapping projects and for interspecific and intraspecific genomic comparisons by techniques such as zoo-FISH.
Journal of Heredity 06/2006; 97(3):303-6. DOI:10.1093/jhered/esj033 · 2.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Expression constructs are subject to position-effects in transgenic assays unless they harbour elements that protect them from negative or positive influences exerted by chromatin at the site of integration. Locus control regions (LCRs) and boundary elements are able to protect from position effects by preventing heterochromatization of linked genes. The LCR in the human beta-globin gene locus is located far upstream of the genes and composed of several erythroid specific DNase I hypersensitive (HS) sites. Previous studies demonstrated that the LCR HS sites act synergistically to confer position-independent and high-level globin gene expression at different integration sites in transgenic mice. Here we show that LCR HS sites 2 and 3, in combination with boundary elements derived from the chicken beta-globin gene locus, confer high-level human beta-globin gene expression in different chromosomal integration sites in transgenic mice. Moreover, we found that the construct is accessible to nucleases and highly expressed when integrated in a centromeric region. These results demonstrate that the combination of enhancer, chromatin opening and boundary activities can establish independent expression units when integrated into chromatin.
Genes to Cells 12/2004; 9(11):1043-53. DOI:10.1111/j.1365-2443.2004.00788.x · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Reports of X chromosome translocations, as primary chromosomal changes associated with hematologic disorders, remain relatively uncommon. Herein, we report the detection, by conventional cytogenetic methods, of a cytogenetically identical t(X;20) in two different patients with hematologic disorders (probable myelodysplasia and polycythemia vera/acute myelocytic leukemia). In both cases, this translocation appeared as the primary clonal chromosome abnormality, with breakpoints occurring in the long arms of both the X chromosome and chromosome 20 (Xq13.1 and 20q13.3, respectively). Further characterization and comparison of the translocation chromosome products of these two cases by use of fluorescence in situ hybridization techniques is also described. Similar previously reported cytogenetically cases and the potential that this specific rearrangement may represent a nonrandom chromosomal finding are discussed.
Cancer Genetics and Cytogenetics 04/2003; 141(2):169-74. DOI:10.1016/S0165-4608(02)00764-1 · 1.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Detailed chromosome studies were conducted for the Florida manatee (Trichechus manatus latirostris) utilizing primary chromosome banding techniques (G- and Q-banding). Digital microscopic imaging methods were employed and a standard G-banded karyotype was constructed for both sexes. Based on chromosome banding patterns and measurements obtained in these studies, a standard karyotype and ideogram are proposed. Characterization of additional cytogenetic features of this species by supplemental chromosome banding techniques, C-banding (constitutive heterochromatin), Ag-NOR staining (nucleolar organizer regions), and DA/DAPI staining, was also performed. These studies provide detailed cytogenetic data for T. manatus latirostris, which could enhance future genetic mapping projects and interspecific and intraspecific genomic comparisons by techniques such as zoo–FISH.
[Show abstract][Hide abstract] ABSTRACT: Previously, we have reported the successful expression of human aldehyde dehydrogenase class-1 (ALDH-1) in K562 leukemia cells using a retroviral vector and demonstrated low expression that resulted in up to three-fold increase in resistance to 4-hydroperoxycyclophosphamide (4-HC), an active derivative to cyclophosphamide. The purpose of this study was to investigate whether in vitro treatment with 4-HC will allow selection of K562 cells expressing higher levels of ALDH-1, and whether these selected cells are more resistant to 4-HC. Stably transfected or transduced K562 cells with retroviral pLXSN vector containing ALDH-1 cDNA (ALDH-1 cells) were treated repeatedly with 4-HC and then allowed to grow to confluence in liquid culture. Subsequently, the resistance to 4-HC of ALDH-1 cells treated once (ALDH-1+) or twice (ALDH-1++) with 4-HC was compared to ALDH-1 cells or wild-type K562 cells (WT cells). The results show significant increase in 4-HC resistance of ALDH-1+ (2- to 16-fold, p < 0.005) over ALDH-1 or WT cells. No difference was detected between ALDH-1+ and ALDH-1++. In addition, higher ALDH-1 mRNA and enzyme activity were found in ALDH-1+ compared to ALDH-1 cells. Southern analysis of DNA extracted from the different experimental groups demonstrated an eight-fold increase in ALDH-1 cDNA in ALDH-1+ versus the ALDH-1 cells. This was confirmed by sequential FISH analysis using biotin labeled pLXSN/ALDH-1 vector. Positive signals consistently localized to the centromeric region of chromosome 9 and the long arm of chromosome 17 were demonstrated only in the ALDH-1+ cells and represented a fusion product of multiple copies of the pLXSN/ALDH-1 vector. In summary, we have demonstrated that in vitro treatment with 4-HC results in the selection of K562 cells with multiple copies of ALDH-1 gene that are clustered in two main integration sites. These cells demonstrate significantly higher resistance to 4-HC when compared to previously untreated cells. Such successful in vitro selection could have significant implications for future cancer gene therapy protocols.
Human Gene Therapy 03/1998; 9(5):611-9. DOI:10.1089/hum.1998.9.5-611 · 3.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We report a jumping translocation involving a donor chromosome 1 long arm in a case of aggressive B-cell non-Hodgkin lymphoma (NHL). Conventional cytogenetic banding studies demonstrated a breakpoint distal to the heterochromatic region of the donor 1q chromosome. Characterization by fluorescence in situ hybridization (FISH) of the jumping translocation demonstrated an apparent telomeric sequence loss of the recipient chromosomes. Additional cytogenetic aberrations, including the t(18;22) translocation associated with non-Hodgkin lymphoma, were also observed in this case. Cytogenetically similar cases of jumping translocations reported in the literature have implicated a preferential involvement of the donor chromosomes' heterochromatic regions and the telomeric regions of the recipient chromosomes. Jumping translocations are still considered rare and their appearance is associated with a poor prognosis. The presence of these specific findings for this case are discussed and compared with those previously reported in other hematologic disorders.
Cancer Genetics and Cytogenetics 11/1997; 98(1):20-7. DOI:10.1016/S0165-4608(96)00409-8 · 1.93 Impact Factor