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Biomedical subjects

F S Wang

Publications and source records attributed to F S Wang.

At least 19 recordsLinked to original sources
PubMed2006

Characterization and stability investigation of cu,zn-superoxide dismutase covalently modified by low molecular weight heparin.

Cu,Zn-superoxide dismutase (SOD) was chemically modified with low molecular weight heparin (LMWH). To characterize the conjugate, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis (native PAGE) with protein staining and polysaccharide staining were employed. The stabilities of the modified enzyme to heat, acid, alkali, and trypsin treatment were also investigated. SDS-PAGE of the conjugate presented two major bands, and native PAGE of the conjugate showed similar banding position with protein staining and polysaccharide staining, which was different from that of the unmodified SOD and LMWH/SOD mixture. Moreover, the conjugate migrated faster with increasing extent of the modification. Enhanced heat stability, acid resistance, alkali resistance, and anti-trypsin stability of the modified enzyme were observed compared with those of the unmodified enzyme. Results of the study suggest that covalent linkage in LMWH-SOD can be effectively characterized by electrophoretic techniques and the chemical modification of SOD with LMWH can enhance the stabilities of the enzyme. In addition, native PAGE with protein staining can be used to evaluate the extent of the modification.

Animals↗
PubMed2005

Persistent shedding of viable SARS-CoV in urine and stool of SARS patients during the convalescent phase.

In order to further the present knowledge of the emerging severe acute respiratory syndrome-associated coronavirus (SARS-CoV), 486 different specimens from 54 patients with a clinical diagnosis of SARS were investigated for the presence of viral RNA, and 314 plasma specimens of 73 patients were examined for IgM and IgG antibodies specific against SARS-CoV using an indirect ELISA. Viral RNA was detectable in 28 of the 54 patients tested. Cumulative data showed that 67 of the 73 SARS patients demonstrated seroconversion by week 5 of illness. In contrast, only 1 of 278 healthy subjects enrolled in the study was found to be positive for the IgG antibody. Coexistence of viral RNA in plasma and specific antibodies was simultaneously observed over three consecutive weeks in two critical cases. In three convalescent patients in particular, cultivable SARS-CoV was detected in stool or urine specimens for longer than 4 weeks (29-36 days). These findings suggest that SARS-CoV may remain viable in the excretions of convalescent patients.

Adult↗
PubMed2005

Acquired but reversible loss of erythrocyte complement receptor 1 (CR1, CD35) and its longitudinal alteration in patients with severe acute respiratory syndrome.

This longitudinal study investigates the change of erythrocyte complement receptor (E-CR1) expression in patients with severe acute respiratory syndrome (SARS). Circulating E-CR1 expression was semiquantified by flow cytometric analyses in 54 SARS patients and in 212 healthy individuals as a control. Since E-CR1 expression is influenced by the genetic polymorphisms in the CR1 gene, a major genetic polymorphism located within intron 27 of the CR1 gene was simultaneously analysed by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). The results showed that the expression level of E-CR1 (referred to as net fluorescence intensity values, NFI) was statistically correlated with the relevant genetic genotypes among the Chinese population including the healthy individuals (NFI: 5.14 +/- 0.82, 3.57 +/- 0.66 and 2.67 +/- 0.32 for HH, HL and LL genotypes, respectively) and SARS patients (NFI: 3.52 +/- 0.91 and 2.63 +/- 0.70 for HH and HL genotypes, respectively). Interestingly, the expression density of E-CR1 was found to fall significantly during the initiation and progressive phases (weeks 1 and 2 after the disease onset) and gradually returned close to normal through their whole convalescent phase (beginning from weeks 2 or 3 to weeks 7 or 8) in SARS patients irrespective CR1 genotype. In conclusion, our findings, at least, suggest that E-CR1 is likely involved in immune pathogenesis of SARS disease.

Adolescent↗
PubMed2005

Cataract surgical coverage and outcome in the Tibet Autonomous Region of China.

BACKGROUND: A recently published, population based survey of the Tibet Autonomous Region (TAR) of China reported on low vision, blindness, and blinding conditions. This paper presents detailed findings from that survey regarding cataract, including prevalence, cataract surgical coverage, surgical outcome, and barriers to use of services. METHODS: The Tibet Eye Care Assessment (TECA) was a prevalence survey of people from randomly selected households from three of the seven provinces of the TAR (Lhoka, Nakchu, and Lingzhr), representing its three main environmental regions. The survey, conducted in 1999 and 2000, assessed visual acuity, cause of vision loss, and eye care services. RESULTS: Among the 15,900 people enumerated, 12,644 were examined (79.6%). Cataract prevalence was 5.2% and 13.8%, for the total population, and those over age 50, respectively. Cataract surgical coverage (vision <6/60) for people age 50 and older (85-90% of cataract blind) was 56% overall, 70% for men and 47% for women. The most common barriers to use of cataract surgical services were distance and cost. In the 216 eyes with cataract surgery, 60% were aphakic and 40% were pseudophakic. Pseudophakic surgery left 19% of eyes blind (<6/60) and an additional 20% of eyes with poor vision (6/24-6/60). Aphakic surgery left 24% of eyes blind and an additional 21% of eyes with poor vision. Even though more women remained blind than men, 28% versus 18% respectively, the different was not statistically significant (p = 0.25). CONCLUSIONS: Cataract surgical coverage was remarkably high despite the difficulty of providing services to such an isolated and sparse population. Cataract surgical outcome was poor for both aphakic and pseudophakic surgery. Two main priorities are improving cataract surgical quality and cataract surgical coverage, particularly for women.

Adult↗
PubMed2004

Detecting human CD34+ and CD34- hematopoietic stem and progenitor cells using a Sysmex automated hematology analyzer.

In clinical medicine, particularly in the newly developing stem cell therapies required to support the practice of regenerative medicine, the measurement of both CD34+ and CD34- hematopoietic stem cells (HSC)/hematopoietic progenitor cells (HPC) is important in obtaining more accurate information about the total HSC/HPC content in various stem/progenitor cell sources. We report the results of an investigation into methods of detecting CD34+ and CD34- HSC/HPC using the immature information (IMI) channel incorporated into the Sysmex XE-2100 and SE-9000 automated hematology analyzers. In this study, CD34+ and CD34- HSC/HPC were separated by immunologic methods and quantified by flow cytometry (FACScan) and IMI channel analysis. In addition, CD34-/CD133+ HSC were prepared by a sequential antibody-based positive selection strategy. These cells appeared in the same area as CD34+ cells in the IMI channel of the automated hematology analyzer. These findings confirmed that an automated hematology analyzer can be used to measure both CD34+ and CD34- HSC. These results may explain the difference in HSC/HPC counts sometimes observed between the automated hematology analyzer and flow cytometric methods for CD34+ measurement. The results of this study demonstrated the potential of automated cell counting methods for measuring HSC content in cellular products for both research and clinical applications.

AC133 Antigen↗
PubMed2003

Genistein directly blocks glycine receptors of rat neurons freshly isolated from the ventral tegmental area.

The effects of tyrosine kinase inhibitors on the glycine-induced current (I(Gly)) were studied in rat neurons freshly isolated from the ventral tegmental area (VTA). Genistein reversibly and concentration-dependently depressed I(Gly), with an IC(50) of 13 microM. Preincubation with genistein had no effect on I(Gly), indicating that genistein is effective only when glycine is bound to the receptor and channels are most likely open. Genistein depressed maximum I(Gly) without significantly changing the EC(50) for glycine. Genistein-induced inhibition of I(Gly) was sensitive to membrane voltage, being greater at positive membrane potentials. A kinetic analysis indicated that genistein lengthens the time constant of I(Gly) activation, but has no effect on deactivation or desensitization. When genistein was rapidly washed out, a transient rebound current probably reflected a faster dissociation of genistein, with respect to glycine. Results of competition experiments suggest that genistein acts on the same region of the glycine receptor as picrotoxin. Daidzein, an analog of genistein that does not act on protein kinases, also inhibited I(Gly). Co-application of lavendustin A, a specific inhibitor of tyrosine kinase, had no effect on I(Gly). Our results extend to neurons isolated from the VTA, the previous finding that genistein directly inhibits glycine receptors of hypothalamic brain slices.

Animals↗
PubMed2003

Blindness and eye diseases in Tibet: findings from a randomised, population based survey.

BACKGROUND: Public health officials of the Tibet Autonomous Region (TAR) of China requested a survey of blindness, eye diseases, and eye care service utilisation to assist the development of a 10 year blindness prevention and treatment plan. The objective of the survey was to determine the prevalence of blindness and visual impairment, as well as cataract surgical coverage and surgical outcome in the TAR. METHODS: The Tibet Eye Care Assessment was a cross sectional prevalence study of three of the seven prefectures (provinces) of the TAR (Lhoka, Nakchu, and Lingzhr) selected to represent its three main environmental regions. The survey sample was selected using a random multistage cluster method. Two teams conducted the survey in a standardised fashion in each prefecture, Lhoka during May and Nakchu during June 1999, and Lingzhr during May 2000. Visual acuity, cause of vision loss, trachoma, and vitamin A deficiency were included in the clinical examination. RESULTS: Among the 15,900 people enumerated, 12,644 were examined for an overall response rate of 79.6%. The crude prevalence of blindness (presenting better eye visual acuity of less than 6/60) was 2.3%; age and sex adjusted blindness prevalence was 1.4% (95% CI 1.3 to 1.5). Visual impairment (better eye presenting visual acuity of 6/24 to 6/60) was found in 10.9% (95% CI 10.5 to 11.2) of the population (age and sex adjusted). Cataract was the primary cause of blindness (50.7%), followed by macular degeneration (12.7%) and corneal opacity (9.7%). CONCLUSION: Blindness is a serious public health problem in Tibet, with prevalence higher than in similar studies in eastern China. As elsewhere in the world, women have an excess burden of blindness compared to men. About 75% of blindness in Tibet can be either prevented or treated. Eye care planning for Tibet must focus on cataract, particularly among women.

Adult↗
PubMed2002

Extracorporeal shock wave promotes growth and differentiation of bone-marrow stromal cells towards osteoprogenitors associated with induction of TGF-beta1.

Extracorporeal shock-wave (ESW) treatment has been shown to be effective in promoting the healing of fractures. We aimed to determine whether ESW could enhance the growth of bone-marrow osteoprogenitor cells. We applied ESW to the left femur of rats 10 mm above the knee at 0.16 mJ/mm2 in a range of between 250 and 2000 impulses. Bone-marrow cells were harvested after ESW for one day and subjected to assessment of colony-forming unit (CFU) granulocytes, monocytes, erythocytes, megakaryocytes (CFU-Mix), CFU-stromal cells (CFU-S) and CFU-osteoprogenitors (CFU-O). We found that the mean value for the CFU-O colonies after treatment with 500 impulses of ESW was 168.2 CFU-O/well (SEM 11.3) compared with 88.2 CFU-O/well (SEM 7.2) in the control group. By contrast, ESW treatment did not affect haematopoiesis as shown by the CFU-Mix (p = 0.557). Treatment with 250 and 500 impulses promoted CFU-O, but not CFU-Mix formations whereas treatment with more than 750 impulses had an inhibiting effect. Treatment with 500 impulses also enhanced the activity of bone alkaline phosphatase in the subculture of CFU-O (p<0.01), indicating a selective promotion of growth of osteoprogenitor cells. Similarly, formation of bone nodules in the long-term culture of bone-marrow osteoprogenitor cells was also significantly enhanced by ESW treatment with 500 impulses. The mean production of TGF-beta1 was 610 pg/ml (SEM 84.6) in culture supernatants from ESW-treated rats compared with 283 pg/ml (SEM 36.8) in the control group. Our findings suggest that optimal treatment with ESW could enhance rat bone-marrow stromal growth and differentiation towards osteoprogenitors presumably by induction of TGF-beta1.

Alkaline Phosphatase↗
PubMed2001

Physical shock wave mediates membrane hyperpolarization and Ras activation for osteogenesis in human bone marrow stromal cells.

Physical shock wave (SW) has shown effectiveness on promotion of bone growth. We have recently demonstrated that SW could promote bone marrow stromal cell differentiation toward osteoprogenitor associated with induction of TGF-beta1. We have further demonstrated that SW-induced membrane hyperpolarization and Ras activation acted an early signal for the osteogenesis in human bone marrow stromal cells. An optimal dose of SW treatment at 0.16 mJ/mm(2) for 500 impulses induced a rapid membrane hyperpolarization in 5 min, activation of Ras in 30 min, and cell proliferation in 2 days. The SW-promoted cell growth was related to osteogenesis as demonstrated by increase of bone alkaline phosphatase activity in 6 days and osteocalcin mRNA expression in 12 days. In support that SW-induced Ras activation mediated osteogenesis of human bone marrow stromal cells, we further demonstrated that transfection of bone marrow stromal cells with a dominant negative Ras mutant (Asn-17 ras(H)) abrogated the SW enhancement of osteogenic transcription factor (CBFA1) activation, osteocalcin mRNA expression, and bone nodule formations. These results suggest that physical SW promotes bone marrow stromal cell differentiation toward osteogenic lineage via membrane hyperpolarization, followed by Ras activation and specific osteogenic transcription factor CBFA1 expression. A link between physical SW and biomembrane perturbation-mediated Ras activation may highlight how noninvasive physical agents could be used to promote fracture healing and to rescue patients with osteoporosis and osteopenic disorders in the future.

Bone Marrow Cells↗
PubMed2001

Neurofilaments consist of distinct populations that can be distinguished by C-terminal phosphorylation, bundling, and axonal transport rate in growing axonal neurites.

We examined the steady-state distribution and axonal transport of neurofilament (NF) subunits within growing axonal neurites of NB2a/d1 cells. Ultrastructural analyses demonstrated a longitudinally oriented "bundle" of closely apposed NFs that was surrounded by more widely spaced individual NFs. NF bundles were recovered during fractionation and could be isolated from individual NFs by sedimentation through sucrose. Immunoreactivity toward the restrictive C-terminal phospho-dependent antibody RT97 was significantly more prominent on bundled than on individual NFs. Microinjected biotinylated NF subunits, GFP-tagged NF subunits expressed after transfection, and radiolabeled endogenous subunits all associated with individual NFs before they associated with bundled NFs. Biotinylated and GFP-tagged NF subunits did not accumulate uniformly along bundled NFs; they initially appeared within the proximal portion of the NF bundle and only subsequently were observed along the entire length of bundled NFs. These findings demonstrate that axonal NFs are not homogeneous but, rather, consist of distinct populations. One of these is characterized by less extensive C-terminal phosphorylation and a relative lack of NF-NF interactions. The other is characterized by more extensive C-terminal NF phosphorylation and increased NF-NF interactions and either undergoes markedly slower axonal transport or does not transport and undergoes turnover via subunit and/or filament exchange with individual NFs. Inhibition of phosphatase activities increased NF-NF interactions within living cells. These findings collectively suggest that C-terminal phosphorylation and NF-NF interactions are responsible for slowing NF axonal transport.

Amoxicillin-Potassium Clavulanate Combination↗
PubMed2001

Selective accumulation of the high molecular weight neurofilament subunit within the distal region of growing axonal neurites.

Axonal maturation in situ is accompanied by the transition of neurofilaments (NFs) comprised of only NF-M and NF-L to those also containing NF-H. Since NF-H participates in interactions of NFs with each other and with other cytoskeletal constituents, its appearance represents a critical event in the stabilization of axons that accompanies their maturation. Whether this transition is effected by replacement of "doublet" NFs with "triplet" NFs, or by incorporation of NF-H into existing doublet NFs is unclear. To address this issue, we examined the distribution of NF subunit immunoreactivity within axonal cytoskeletons of differentiated NB2a/d1 cell and DRG neurons between days 3-7 of outgrowth. Endogenous immunoreactivity either declined in a proximal-distal gradient or was relatively uniform along axons. This distribution was paralleled by microinjected biotinylated NF-L. By contrast, biotinylated NF-H displayed a bipolar distribution, with immunoreactivity concentrated within the proximal- and distal-most axonal regions. Proximal biotinylated NF-H accumulation paralleled that of endogenous NF immunoreactivity; however, distal-most biotinylated NF-H accumulation dramatically exceeded that of endogenous NFs and microinjected NF-L. This phenomenon was not due to co-polymerization of biotin-H with vimentin or alpha-internexin. This phenomenon declined with continued time in culture. These data suggest that NF-H can incorporate into existing cytoskeletal structures, and therefore suggest that this mechanism accounts for at least a portion of the accumulation of triplet NFs during axonal maturation. Selective NF-H accumulation into existing cytoskeletal structures within the distal-most region may provide de novo cytoskeletal stability for continued axon extension and/or stabilization.

Animals↗
PubMed2001

Dysfunction of peripheral blood dendritic cells from patients with chronic hepatitis B virus infection.

AIM: To identify the property of dendritic cells (DCs) of peripheral blood monocytes (PBMC) in patients with chronic HBV infection. METHODS: Twenty patients with persistent HBV infection were included in this study, 10 healthy subjects being used as a control group. The peripheral blood mononuclear cells (PBMC) of T cell-depleted populations were incubated and induced into mature dendritic cells in the RPMI-1640 medium in the presence of cytokines GM-CSF, IL-4, FLt-3,TNF-alpha and 100mL.L(-1 )of fetal calf serum for a total of 10-12 days. The expressions of surface markers on DCs were evaluated using flow cytometric analysis. ELISA method was used to determine the cytokine levels of interleukin-12 (IL-12) and IL-10 in the supernatant produced by DCs. For detection of the stimulatory capacity of DCs to T cell proliferation, mytomycin C-treated DC were incubated with allogenic T cells. RESULTS: A typical morphology of mature DCs from healthy subjects and HBV-infected patients was induced in in vitro incubation, but the proliferation ability and cellular number of DCs from HBV-infected patients significantly decreased compared with healthy individuals. In particular, the expression levels of HLA-DR, CD80 (B7-1) and CD86 (B7-2) on DC surface from patients were also lower than that from healthy individuals (0.46 vs 0.92 for HLA-DR, 0.44 vs 0.88 for CD80 and 0.44 vs 0.84 for CD86,P<0.05). The stimulatory capacity and production of IL-12 of DCs from patients in allogenic mixed lymphocyte reaction (AMLR) significantly decreased, but the production level of nitric oxide (NO) by DCs simultaneously increased compared with healthy subjects (86 +/- 15 vs 170 +/- 22 micromol.L(-1), P <0.05). CONCLUSION: The patients with chronic HBV infection have the defective function and immature phenotype of dendritic cells, which may be associated with the inability of efficient presentation of HBV antigens to host immune system for the clearance of HBV.

Adolescent↗
PubMed2000

Flow cytometric method for enumeration and classification of reactive immature granulocyte populations.

We developed a flow cytometric method for the enumeration and classification of nonmalignant immature granulocytes (IG). In this study, IG are defined as most immature (IG stage 1: promyelocytes and myelocytes) and as more mature (IG stage 2: metamyelocytes). Blood specimens from 46 patients with documented infectious or inflammatory disease and known presence of IG (by routine manual microscopy) were analyzed. For a reference manual differential count, we used a 400 white blood cell (WBC) differential and separated granulocytes into promyelocytes and myelocytes combined, metamyelocytes, and included band cells in the mature, segmented neutrophil population. The flow cytometric method is based on three-color staining of whole, anticoagulated blood with CD45-PerCP, CD16-FITC, and CD11b-PE-labeled monoclonal antibodies and a three-step gating procedure. The flow cytometric results were confirmed by cell sorting and microscopic evaluation of the sorted cells. A total of 10,000 events, excluding debris, were recorded per specimen and IG stage 1 (CD16-/CD11b-), IG stage 2 (CD16-/CD11b+), and mature neutrophils (CD16+/CD11b+) were categorized. Regression and correlation between flow cytometric IG and the manual differential showed y = 1.34x + 0.95, r(2) = 0.86 for IG stages 1 and 2 combined versus promyelocytes, myelocytes, and metamyelocytes. For IG stage 1 versus microscopic counts of promyelocytes and myelocytes, the results were y = 1.53x + 1.24, r(2) = 0.76; for IG stage 2 versus manual metamyelocyte count, y = 0.77x + 0.21, r(2) = 0.58. Reproducibility of the flow cytometric method showed a coefficient of variation (CV) of 6.8% for all IG combined compared with a CV of 50.2% for manual differential IG count (based on a routine 100 WBC count). Samples were found stable at least 12 h at 25 degrees C and at least 48 h at 4 degrees C for flow cytometry. After staining and lysing, the sample was stable for at least 120 min at room temperature. We analyzed samples from patients with myelodysplastic and myeloproliferative disease separately. We found that CD16- mature neutrophils falsely elevated the flow cytometric IG count. Similar results were obtained in blood from patients treated with granulocyte-colony stimulating factor (G-CSF). Although this restricts the use of the method somewhat, we believe that this flow cytometric method is useful for enumerating reactive IG, as well as for evaluating automated methods for IG identification by hematology analyzers.

Flow Cytometry↗
PubMed2000

[Construction and bioassay of secondary recombinant baculovirus with two foreign gene].

Plasmid pAcLEneo which bears neomycin gene derived by the baculovirus IE1 promotor was digested and the gene was harvested. A transfer vector pAc34DZ2 in which the polyhedrin envelope gene has been inactivated by insertion of expression cassette of PIE1/neo was constructed by inserting the neo cassette into the SacI site of plasmid pAc34DZ1. We have constructed the polyhedrin positive recombinant virus (I) vAcPhBtT which was able to express Bt truncated endotoxin gene. In order to improve the insecticide efficiency of recombinant virus (I), an anti-neomycin recombinant virus (II) vAcPhBtTPE- was obtained by second co-transfection into the Sf9 cells with pAc34DZ2 and recombinant virus (I) DNA. Southern blot and SDS-PAGE analysis indicated that the recombinant virus (II) was still able to express the 80 kD Bt truncated delta-endotoxin, but did not express the 34 kD polyhedrin envelope protein. The recombinant virus (II) without envelope released virion faster than recombinant virus (I) after alkaline lysis. Bioassay was carried out using Spodoera exigua. LC50 of the recombinant virus (II) was about half of wild type virus and LT50 reduced about two days.

Animals↗
PubMed1999

The neural cell adhesion molecules L1 and NCAM-180 act in different steps of neurite outgrowth.

The formation of neurocircuitry depends on the control of neurite outgrowth that, in turn, can be divided into two processes: nerve growth cone protrusion and neurite extension. It has long been known that the neural cell adhesion molecules L1 and NCAM-180 promote neurite outgrowth, but how they function in growth cones is unclear. We addressed the roles of L1 and NCAM-180 in neurite outgrowth by using microscale chromophore-assisted laser inactivation (micro-CALI) of these proteins to perturb their functions at precise times in single growth cones of embryonic chick dorsal root ganglion neurons grown in culture. Micro-CALI of L1 causes neurite retraction after a 10 min lag period but does not affect growth cone protrusion. In contrast, micro-CALI of NCAM-180 causes rapid growth cone retraction but does not affect neurite extension. The simultaneous inactivation of both these molecules resulted in both distinct effects that were segregated in time. The behavior of growth cones after these micro-CALI treatments resemble the drug-induced perturbation of microtubules for L1 and F-actin for NCAM-180. These findings suggest distinct roles in the growth cone for L1 and NCAM-180 in different steps of neurite outgrowth: L1 functions in neurite extension,whereas NCAM-180 functions in growth cone protrusion.

Animals↗
PubMed1999

Retrovirus-mediated transfer of anti-MDR1 hammerhead ribozymes into multidrug-resistant human leukemia cells: screening for effective target sites.

One of the underlying mechanisms of multidrug resistance (MDR) is cellular over-production of P-glycoprotein (P-gp), which acts as a drug efflux pump. P-gp is encoded by a small group of related genes termed MDR; only MDR1 is known to confer drug resistance. To overcome P-gp-mediated drug resistance, we have developed two anti-MDR1 hammerhead ribozymes driven by the beta-actin promoter. Upon transduction of the ribozymes into MDR cells, vincristine resistance was decreased. These two ribozymes were constructed, which showed different cleavage activities. In this study, to determine suitable target sites for the anti-MDR1 ribozyme, the exon 1b-intron 1 boundary, the translation-initiation site, the intron 1-exon 2 boundary and the exon 2-intron 2 boundary, codons 179 and 196 of the MDR1 gene were selected as candidates. To improve the ribozyme activity, a retroviral vector containing RNA polymerase III promoter was used. Stable retrovirus producer cells were generated by transfecting the retroviral vector plasmids carrying the ribozyme into the packaging cell line. Retroviral vector transduction of human leukemia cell lines expressing MDR1 was accomplished by co-culturing these with virus producer cells. Stably transduced cells were selected by G418 and pooled to determine the efficacy of each ribozyme. These ribozyme-transduced cells became vincristine-sensitive concomitant with the decreases in MDR1 expression, P-gp amount and drug efflux pump function. Among the ribozymes tested, the anti-MDR1 ribozyme against the translation-initiation site exhibited the strongest efficacy. This retrovirus-mediated transfer of anti-MDR1 ribozyme may be applicable to the treatment of MDR cells as a specific means to reverse resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Retrovirus-mediated transfer of anti-MDR1 ribozymes fully restores chemosensitivity of P-glycoprotein-expressing human lymphoma cells.

Development of multidrug resistance (MDR) is the major obstacle to successful cancer chemotherapy. We have developed Daudi human lymphoma cells that are 20-fold more resistant than the parent cell line to vincristine (VCR) by infecting cells with pHaMDR1/A retroviral vector (Daudi/MDR20). Three DNA sequences of anti-MDR1 hammerhead ribozymes (Rzs), one cleaving codon 196 of MDR1 mRNA (196MDR1-Rz), the second a stem II base-modified (U9-->Gg, U13-->A13, G14-->A14, A18-->C18) Rz against codon 196 (196MDR1-sRz), and the third a stem II base-modified Rz directed against the -6 approximately -4 GUC sequence of the translation initiation site of the MDR1 mRNA (iMDR1-sRz), were synthesized and cloned into the retroviral vector N2A+tRNAiMet downstream of the RNA polymerase III promoter and adjacent to a tRNA gene sequence, forming the constructs N2A+tRNAiMet-196MDR1-Rz, N2A+tRNAiMet-196MDR1-sRz, and N2A+tRNAiMet-iMDR1-sRz. The three constructs were transfected into GP+envAM 12 cells for packaging the retroviral vectors. The supernatants containing the packaged retrovirus in high titers (1.1-2.5 X 10(5) CFU/ml as determined by infection of NIH 3T3 cells) were used to infect Daudi/MDR20 cells. The iMDR1-sRz- and 196MDR1-sRz-transduced Daudi/MDR20 cells completely restored chemosensitivity to VCR and doxorubicin, and were accompanied by blocked expression of MDR1 mRNA and P-glycoprotein as well as overexpression of anti-MDR1 Rz. In a cell-free system, the chimeric tRNA-sRz molecules were more stable and had more efficient catalytic activities than the corresponding naked Rz molecules. The stem II base-modified Rz were also more stable and efficient in catalytic activities than the unmodified Rz molecules. The base modification in the Rz stem II structure and the development of chimeric tRNA-Rz molecules were identified to enhance the cleavage efficacy. The combination of these two factors, together with the use of a retroviral vector, appear to have contributed to the complete reversal of MDR.

ATP Binding Cassette Transporter, Subfamily B↗