Biomedical subjects

L C Harris

Publications and source records attributed to L C Harris.

At least 37 records · Page 2Linked to original sources

PubMed1998

Bax is an important determinant of chemosensitivity in pediatric tumor cell lines independent of Bcl-2 expression and p53 status.

Susceptibility of a tumor cell to undergo chemotherapy-induced apoptosis appears to be dependent upon the balance of proapoptotic and survival factors that are expressed within any given cell. We have chosen to evaluate how expression of several of these proteins influences chemosensitivity of a panel of 10 pediatric tumor cell lines chosen from three tumor histiotypes: neuroblastoma, rhabdomyosarcoma, and pediatric glial tumors. The proteins evaluated were p53 and six members of the Bax/Bcl-2 family: three proapoptotic proteins (Bax, Bak, and Bcl-xS) and three survival factors (Bcl-2, Bcl-xL, and Mcl-1). We investigated whether there was any relationship between endogenous expression of these proteins and chemosensitivity (or resistance) to three chemotherapeutic agents that directly damage DNA (doxorubicin, actinomycin D, and topotecan) and a mitotic spindle poison (vincristine). Even though exogenous overexpression of wild-type p53 has been associated with a chemosensitive phenotype in several model systems we demonstrated no such relationship in these studies. In addition, expression levels of Bcl-2, Bcl-xL, Bcl-xS, Bak, or Mcl-1 did not correlate with sensitivity or resistance to the four drugs. However, there was a statistically significant correlation between endogenous levels of Bax protein and sensitivity to both doxorubicin and actinomycin D. We conclude that even though many proteins such as p53 and Bcl-2 have been shown to influence drug response when exogenously overexpressed in model systems, in unmodified cell lines endogenous protein levels may not generate the same results. We have demonstrated that endogenous Bax expression was the only protein found to be associated with chemosensitivity across the three different tumor histiotypes and propose that analysis of Bax may be a more useful prognostic indicator for tumor response to therapy than either p53 or Bcl-2.

Animals↗

PubMed1997

Cytoplasmic sequestration of an O6-methylguanine-DNA methyltransferase enhancer binding protein in DNA repair-deficient human cells.

O6-Methylguanine-DNA methyltransferase (MGMT), an enzyme that repairs adducts at O6 of guanine in DNA, is a major determinant of susceptibility to simple methylating carcinogens or of tumor response to anticancer chloroethylating drugs. To investigate the mechanisms underlying cellular expression of this DNA repair enzyme, we focused on the role of a 59-bp enhancer of the human MGMT gene in the regulation of its expression. By using chloramphenicol acetyltransferase reporter assays, we found that the enhancer activity, which was present in both MGMT-expressing (Mer+) and -deficient (Mer-) cells, correlated with the endogenous MGMT activity in Mer+ cell lines. Band-shift assays and deletion analysis of the 59-bp sequence defined a minimal 9-mer cis element (5'-CTGGGTCGC-3') for specific trans factor binding. The MGMT enhancer binding protein (MEBP), 45 kDa by Southwestern blot analysis, was present in the nuclei of all Mer+ cells tested but was apparently restricted to the cytoplasm of Mer- cells. We conclude that the MEBP-enhancer interaction plays an important role in regulating constitutive MGMT expression in Mer+ cells and that MEBP exclusion from the nucleus may account for the down-regulation of MGMT in Mer- cells.

Base Sequence↗

PubMed1997

The risk of peripheral vein phlebitis associated with chlorhexidine-coated catheters: a randomized, double-blind trial.

OBJECTIVE: To evaluate the risk of phlebitis associated with chlorhexidine-coated polyurethane catheters in peripheral veins. DESIGN: A randomized, double-blinded trial comparing chlorhexidine-coated polyurethane catheters with uncoated polyurethane catheters. SETTING: A university hospital. PATIENTS: Adult medicine and surgery patients. INTERVENTIONS: Certified registered nurse anesthetists or an infusion team consisting of nurses and physicians inserted the catheters. Catheter insertion sites were scored twice daily for evidence of phlebitis. At the time catheters were removed, a quantitative blood culture was performed, and catheters were sonicated for quantitative culture. RESULTS: Of 221 evaluable catheters, phlebitis developed in 18 (17%) of 105 coated catheters, compared to 27 (23%) of 116 uncoated catheters (relative risk [RR], 0.74; 95% confidence interval [CI95], 0.43-1.26; P = .32). By survival analysis, chlorhexidine-coated catheters had a lower risk of phlebitis during the first 3 days (P = .06), but not when all catheters were considered in both patient groups (P = .31). In the absence of catheter colonization, the incidence of phlebitis was 21% (16/76) and 24% (20/86) for coated and uncoated catheters, respectively (P = .85), whereas in the presence of catheter colonization, the incidence of phlebitis was 14% (1/7) and 80% (4/5) for coated and uncoated catheters, respectively (RR, 0.18; CI95, 0.03-1.15; P = .07). CONCLUSION: The risk of phlebitis in the presence of catheter colonization was 82% lower for chlorhexidine-coated polyurethane catheters compared to otherwise identical uncoated catheters.

Adult↗

PubMed1997

Retroviral transfer of a bacterial alkyltransferase gene (ada) into human bone marrow cells protects against O6-benzylguanine plus 1, 3-bis(2-chloroethyl)-1-nitrosourea cytotoxicity.

The antitumor activity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) is limited by the O6-alkylguanine-DNA alkyltransferase (ATase) in tumor cells and by delayed myelosuppression. Inactivation of neoplastic ATase by O6-benzylguanine (BG) improves the therapeutic index for BCNU. We have demonstrated previously that BG + BCNU-induced myelosuppression in mice is reduced by expression of the BG-resistant ATase ada in murine bone marrow. We have now generated an amphotropic retrovirus containing the ada gene and tested the effectiveness of ada expression in preventing BG + BCNU cytotoxicity in human hematopoietic progenitor cells. A retroviral producer clone with a biological titer of 6.5 x 10(4) colony-forming units/ml and 4.4 pmol ATase/mg protein was used for transduction of bone marrow. Cocultivation of these ada producer cells with progenitor cells from six normal individuals resulted in 1.9-3. 9-fold protection against BG + BCNU-induced cytotoxicity in committed progenitor cell assays. Furthermore, this cytoprotective effect was associated with a high transduction efficiency (40%) and a 2-fold increase of ATase activity in the surviving committed progenitor cell colonies. These data provide a basis for testing the clinical effectiveness of retroviral ada gene transfer into hematopoietic cells to increase the therapeutic index of BG + BCNU.

3T3 Cells↗

PubMed1996

Wild-type p53 suppresses transcription of the human O6-methylguanine-DNA methyltransferase gene.

High-level expression of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) correlates with cellular resistance to the chloroethylnitrosourea (CENU) class of alkylating agents. Consequently, tumors expressing low levels of MGMT are sensitive to CENU chemotherapy, and any mechanism that can be used to reduce MGMT levels could sensitize resistant tumors. We have demonstrated that transient transfection of wild-type, but not mutant, p53 protein into a p53-null cell line, Saos-2, suppresses MGMT promoter activity in a reporter gene system. In addition, following a 24-h transduction of IMR90 fibroblasts with a wild-type p53-adenoviral vector, endogenous MGMT protein is down-regulated and is no longer detectable 5 days following infection. Because p53 is inducible by ionizing radiation, we propose that existing cancer therapy regimens that combine radiotherapy with CENU chemotherapy may be improved by altering scheduling and allowing enough time between the two therapies for the relatively stable MGMT protein to degrade.

Down-Regulation↗

PubMed1996

Oxygen administration during transport and recovery after outpatient surgery does not prevent episodic arterial desaturation.

STUDY OBJECTIVE: To compare the efficacy of two different oxygen (O2) delivery systems in preventing episodic arterial desaturation in the immediate postoperative period. STUDY DESIGN: Randomized, prospective, nonblinded comparison in patients. SETTING: Operating room and postanesthesia care unit (PACU) of a university outpatient surgery center. PATIENTS: 100 ASA status I and II adults, male and female, age greater than 18 years, undergoing outpatient surgical procedures not involving the upper airway. INTERVENTIONS: Group I received supplemental O2 administered by bag-valve-mask during transport, followed by 40% face shield in the PACU. Group 2 received supplemental O2 by nasal cannula at 4 L/min both during transport and during PACU stay. MEASUREMENTS AND MAIN RESULTS: Arterial O2 saturation (SpO2) was collected by computer from a recording pulse oximeter at 15-second intervals beginning before extubation and continuing until O2 administration was discontinued in PACU. Neither mode of therapy was successful in completely eliminating arterial desaturation defined as SpO2 less than 90%, nor was there any difference in efficacy between the two treatment groups. Group 1 had 8 patients desaturate on 9 different occasions (5 times during transport, 4 times in PACU). Group 2 had 5 patients desaturate on 9 different occasions (4 times during transport, 5 times in PACU). CONCLUSION: Routine O2 administration during transport and PACU stay did not abolish episodic desaturation, even in healthy patients undergoing minor surgical procedures. Given the marked difference in acquisition cost, it would appear that O2 administration by nasal cannula is a more cost-effective alternative for routine postoperative O2 administration in certain groups of patients undergoing general anesthesia for outpatient surgery.

Adult↗

PubMed1996

Changes in O6-methylguanine-DNA methyltransferase expression during immortalization of cloned human fibroblasts.

Suppressed expression of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT), characterized as the Mer- phenotype, occurs only in malignant or transformed cell lines. To investigate the relationship between the transformation process and loss of MGMT expression, we derived 20 cloned lines of IMR90 normal fibroblasts transfected with the plasmid pSV3neo expressing the SV40 large-T antigen. Of the five lines that were grown until crisis phase, four emerged as continuously proliferating immortal lines. Of these, only one retained MGMT, the other three having become Mer-. In every case the loss of MGMT coincided with the final phase of immortalization following crisis. Because these were cloned cell lines it is clear that the phenotypic change to Mer- is not merely due to selection of a Mer- cell from the initial population, but must involve a cellular change in MGMT regulation. It is not clear if increased mutation rate associated with loss of MGMT results in increased frequency of an immortalization event or if an immortalization event, such as telomere disruption, results in MGMT suppression. In addition, we have shown that, consistent with previous observations, both hypermethylation in promoter sequences and hypomethylation of downstream sequences in the body of the gene were closely associated with loss of MGMT expression. These studies also illustrate the utility of these new cloned cell lines for characterizing molecular events associated with transformation and immortalization.

Antigens, Polyomavirus Transforming↗

PubMed1995

Retroviral transfer of a bacterial alkyltransferase gene into murine bone marrow protects against chloroethylnitrosourea cytotoxicity.

The chloroethylnitrosoureas (CENUs) are important antineoplastic drugs for which clinical utility has been restricted by the development of severe delayed myelosuppression in most patients. To investigate the potential of DNA repair proteins to reduce bone marrow sensitivity to the CENUs, we transferred the Escherichia coli ada gene, which encodes a Mr 39,000 O6-alkylguanine-DNA alkyltransferase (ATase), into murine bone marrow cells by the use of a high-titer ecotropic retrovirus. The ada-encoded ATase is resistant to O6-benzylguanine (O6-BG), a potent inhibitor of the mammalian ATases, thus affording the bone marrow an additional level of protection against CENUs. In methylcellulose cultures, ada-infected hematopoietic progenitor cells were twice as resistant as uninfected cells to the toxic effects of 1, 3-bis(2-chloroethyl)-1-nitrosourea (BCNU) following treatment with O6-BG. Although showing no obvious protective effects against leukopenia, overexpression of the bacterial ATase activity reduced the severity of anemia and thrombocytopenia in mice treated with O6-BG and BCNU. These effects, which were maximal at a BCNU dose of 12.5 mg/kg, were associated with improved survival when BCNU was given at this dose. At lower BCNU doses cytotoxicity was limited in both transduced and control mice, and at higher doses the protective effect was saturated due to cytotoxicity. These results suggest that ada gene therapy may be a feasible approach to amelioration of delayed myelosuppression following O6-BG plus CENU combination chemotherapy.

Animals↗

PubMed1994

Identification of a 59 bp enhancer located at the first exon/intron boundary of the human O6-methylguanine DNA methyltransferase gene.

The DNA repair enzyme, O6-methylguanine DNA methyltransferase (MGMT) is responsible for repair of damage induced by alkylating agents that produce adducts at O6-guanine in DNA. Although the MGMT gene promoter has housekeeping gene promoter characteristics, unlike these genes which are expressed at a constant level, MGMT transcriptional activity varies between cell types. During an attempt to identify regions of the MGMT regulatory sequence sensitive to variations in transcription factors between cell types, we have identified a 59 bp enhancer which is required for efficient MGMT promoter function. This fragment produced increased transcriptional activity in reporter gene constructs containing either the MGMT or UMP-synthase promoter when transfected into either of two cell lines; it seems therefore that this enhancer may interact with relatively common trans-acting factors. Functional activity is only detected when the enhancer is in 'cis' with respect to the promoter, suggesting that complexes are formed between proteins bound to the enhancer and promoter sequences. We propose that the enhancer-binding protein may be a novel transcription factor since there are no obvious consensus sequences within the 59 bp sequence for known DNA-binding proteins.

Base Sequence↗

PubMed1994

In vitro methylation of the human O6-methylguanine-DNA methyltransferase promoter reduces transcription.

Approx. 20% of human tumor cell lines (termed Mer-) are deficient in the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT; E.C.2.1.1.63). Such cells possess the MGMT gene and promoter sequences but have virtually no mRNA or protein. Cytosine methylation of gene sequences has been proposed as a mechanism by which MGMT could be suppressed in Mer- cells; however, the experimental evidence does not uniformly support this idea. We therefore investigated the effect of in vitro methylation of the MGMT promoter in a reporter gene construct transfected into cultured human cells. DNA methylation by HpaII or HhaI methylases suppressed the activity of the promoter, although the effect was not absolute. The occurrence of partial intracellular demethylation of promoter sequences may account for the incomplete inhibition of transcription. A model that attempts to reconcile the opposing views on the role of cytosine methylation in MGMT gene expression is presented.

Humans↗

PubMed1993

Ribozyme-mediated modulation of human O6-methylguanine-DNA methyltransferase expression.

A synthetic oligonucleotide containing ribozyme sequences targeted to the 5' region of the human O6-methylguanine-DNA methyltransferase (MGMT) mRNA has been constructed. This ribozyme demonstrates cleavage activity in vitro in the presence of Mg2+. To determine whether this ribozyme can function in vivo, HeLa CCL2 cells were transfected with a mammalian expression vector containing the ribozyme sequence. Following selection and expansion of individual transfectants, a stable clone was isolated that lacks both MGMT mRNA and protein. Molecular analysis of this cell line indicates that in vivo cleavage of MGMT mRNA is responsible for the lack of MGMT activity.

Base Sequence↗

PubMed1993

Expression in mammalian cells of the Escherichia coli O6 alkylguanine-DNA-alkyltransferase gene ogt reduces the toxicity of alkylnitrosoureas.

V79 Chinese hamster cells expressing either the O6-alkylguanine-DNA-alkyltransferase (ATase) encoded by the E. coli ogt gene or a truncated version of the E. coli ada gene have been exposed to various alkylnitrosoureas to investigate the contribution of ATase repairable lesions to the toxicity of these compounds. Both ATases are able to repair O6-alkylguanine (O6-AlkG) and O4-alkylthymine (O4-AlkT) but the ogt ATase is more efficient in the repair of O4-methylthymine (O4-MeT) and higher alkyl derivatives of O6-AlkG than is the ada ATase. Expression of the ogt ATase provided greater protection against the toxic effects of the alkylating agents then the ada ATase particularly with N-ethyl-N-nitrosourea (ENU) and N-butyl-N-nitrosourea (BNU) to which the ada ATase expressing cells were as sensitive as parent vector transfected cells. Although ogt was expressed at slightly higher levels than the truncated ada in the transfected cells, this could not account for the differential protection observed. For-N-methyl-N-nitrosourea (MNU) the increased protection in ogt-transfected cells is consistent with O4-MeT acting as a toxic lesion. For the longer chain alkylating agents and chloroethylating agents, the protection afforded by the ogt protein may be a consequence of the more efficient repair of O6-AlkG, O4-AlkT or both of these lesions in comparison with the ada-encoded ATase.

Animals↗

PubMed1993

Performance characteristics and interanalyzer variability of PO2 measurements using tonometered human blood.

We performed a side-by-side comparison of the ability of four blood gas analyzers (IL-1312, Corning-178, AVL-995, and ABL-330) to measure PO2 across a wide range under controlled laboratory conditions. Samples of fresh whole human blood, tonometered with analytic quality gas, were prepared with partial pressures of oxygen from 0 to 283 mm Hg. Fifteen determinations were made at 16 levels of tonometric PO2 (tPO2) on each of the four blood gas analyzers. The bias, precision, and root mean squared error (RMSE) of the PO2 measurement relative to tPO2 were determined for each analyzer at each tPO2 level. Mean bias and precision across the range tested were 2.78 +/- 1.29 mm Hg (IL), -0.35 +/- 1.91 (Corning), 2.14 +/- 1.43 (AVL), and 3.00 +/- 1.47 (ABL). RMSE was 3.28, 2.61, 3.57, and 2.41 for IL, AVL, ABL, and Corning, respectively. Percent RMSE (RMSE/tPO2 x 100%), ranged from 0.9% (AVL at 75 mm Hg PO2 and IL at 283 mm Hg tPO2) to 9.1% (IL at 29 mm Hg tPO2). Three analyzers (AVL, ABL, and Corning) showed a statistically significant (p < 0.0001) correlation between RMSE and tPO2, and no correlation between percent RMSE and tPO2. This demonstrates that, for these instruments, accuracy is a function of the magnitude of the tPO2 value. IL did not show a significant correlation between RMSE and tPO2 but did demonstrate a significant negative correlation (r = -0.78, p > 0.001) between percent RMSE and tPO2, indicating that, for this analyzer, accuracy is not a function of tPO2. The differences in PO2 measurements between pairs of analyzers were also examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Arteries↗

PubMed1992

A comparison of human O6-methylguanine-DNA methyltransferase promoter activity in Mer+ and Mer- cells.

The activity of the human O6-methylguanine-DNA methyltransferase (MGMT) gene promoter was determined in eight human cell lines by measuring chloramphenicol acetyltransferase activity in a reporter gene system. MGMT promoter activities in cells that do not express MGMT (Mer-) fell within the range of activities seen in cells that do express MGMT (Mer+). The promoter region contains 11 potential binding sites for the transcription factor Sp1, but no correlation was seen between cellular Sp1 protein and MGMT promoter chloramphenicol acetyltransferase activity. Because Mer- cells are not deficient in the factors needed for transcription of MGMT, we suggest that at least two mechanisms regulate MGMT expression. One suppresses MGMT mRNA and protein in Mer- cells, and another regulates the levels of constitutive expression in Mer+ cells. Sp1 is not a limiting factor in MGMT expression.

Blotting, Western↗

PubMed1992

Site directed mutagenesis of two cysteine residues in the E. coli ogt O6-alkylguanine DNA alkyltransferase protein.

The E. coli ogt O6-alkylguanine-DNA alkyltransferase has two cysteine residues positioned identically with respect to cysteines in the E. coli ada O6-alkylguanine-DNA alkyltransferase. In order to assess their function, these residues were each substituted by a glycine to generate altered forms of the ogt protein. Mutagenesis of cysteine-139, located within a 'PCHRV' region of homology, eliminated functional activity confirming that this residue is the methyl-accepting cysteine in the active site of the protein. Substitution of cysteine 102 within the sequence 'LRTIPCG' had little effect on the ogt protein activity demonstrating that this cysteine is not directly involved with the transfer of O6-methylguanine adducts.

Amino Acid Sequence↗

PubMed1992

A comparison of three pulmonary artery oximetry catheters in intensive care unit patients.

OBJECTIVE: To compare the clinical performance of three pulmonary artery oximetry catheters (Oximetrix 3, SAT-2, and HEMOPRO2) in intensive care unit (ICU) patients. DESIGN: Unblinded comparison of performance over 24 h using an IL-282 CO-oximeter as a criterion standard. SETTING: Multispecialty adult ICU at a university teaching hospital. PATIENTS: Thirty critically ill patients selected from those requiring hemodynamic monitoring for medical management. MEASUREMENTS AND MAIN RESULTS: By all measures, performance of the Oximetrix 3 and SAT-2 systems were comparable; bias +/- precision were -1.98 +/- 3.07 and +1.80 +/- 3.49, respectively, vs -2.28 +/- 5.24 for the HEMOPRO2. The Oximetrix 3 and SAT-2 systems demonstrated consistent performance over the range of saturations tested, though Oximetrix 3 tended to underestimate and SAT-2 tended to overestimate the CO-oximeter value. The HEMOPRO2 underestimated the CO-oximetry-derived saturation, although this was not constant across the range of values tested. The 95 percent confidence limits based on intrasubject variability were similar (+/- 4.59, +/- 5.66, and +/- 6.56 for the Oximetrix 3, SAT-2, and HEMOPRO2, respectively); however, the 95 percent confidence limits based on total variability, while similar for Oximetrix 3 (+/- 6.03) and SAT-2 (+/- 6.86), were larger for the HEMOPRO2 (+/- 10.30). The expected SD was similar for the three systems (2.03, 2.50, and 2.90 for the Oximetrix 3, SAT-2, and HEMOPRO2 systems, respectively). None of the systems equaled or exceeded (p greater than 0.05) the manufacturers' published specifications, which, in all cases, are listed as +/- 2 percent (saturation; 1 SD) when compared with bench oximetry. CONCLUSIONS: Although each system measures mixed venous oxygen saturation, the Oximetrix 3 and SAT-2 systems demonstrate closer agreement with CO-oximetry. However, none of these catheters provided statistically significant evidence that they would perform within +/- 2 percent of CO-oximetry. As a continuous monitor used to detect changes or trends, any of the three may be acceptable.

Calibration↗

PubMed1992

Cytosine methylation and suppression of O6-methylguanine-DNA methyltransferase expression in human rhabdomyosarcoma cell lines and xenografts.

Human tumor cell lines that do not express O6-methylguanine-DNA methyltransferase (MGMT) in detectable quantities (Mer-) are hypersensitive to the effects of O6-guanine-alkylating agents. Because the Mer- phenotype enhances tumor response to such agents, we investigated possible mechanisms involved in regulation of MGMT expression in a panel of Mer+ and Mer- pediatric rhabdomyosarcoma xenograft and cell lines. All Mer- cell and xenograft lines lacked not only MGMT activity but also the protein and mRNA as well, suggesting that its expression is transcriptionally regulated. Transfection of Mer+ and Mer- rhabdomyosarcoma cell lines with MGMT gene promoter-CAT constructs yielded similar levels of CAT expression, indicating that Mer- cells possessed the necessary factors to support transcription. Methylation in the 5'-untranslated region of the MGMT gene was assayed by Southern analysis using methylation-sensitive restriction enzymes. Digestion with HpaII and its methylation-insensitive isoschizomer, MspI, revealed little overall correlation between methylation and MGMT expression. However, methylation in a single SmaI site at position-69 was observed in all MGMT deficient lines but not in any MGMT expressing lines. These results suggest that methylation of specific cytosines in the MGMT promoter may play a role in suppressing its expression, as well as being a potentially useful marker for the Mer- phenotype.

Animals↗

PubMed1991

Characterization of the promoter region of the human O6-methylguanine-DNA methyltransferase gene.

O6-methylguanine-DNA methyltransferase (MGMT) is a ubiquitous protein responsible for repair of O6-alkylguanine, a mutagenic, carcinogenic and toxic lesion. To characterize the elements responsible for the regulation of the MGMT gene, a 2.6 kb Sstl fragment isolated from a genomic clone, was shown to contain 5' flanking sequences of the gene. The promoter activity of this fragment as well as various subfragments were tested in NIH 3T3 mouse fibroblasts by transient expression of the bacterial chloramphenicol acetyltransferase (CAT) gene linked to these fragments. Maximal promoter activity was observed in a 1.2 kb 3' terminal fragment, which contains the first untranslated exon. The transcription initiation site was identified in this fragment by primer extension and S1 mapping. Sequence analysis of this fragment showed the absence of TATA and CAAT boxes but an abundance of extremely GC-rich sequences, including ten GC hexanucleotide motifs 5'CCGCCC. Reduced CAT expression with the minimal promoter sequence suggests the presence of multiple regulatory elements.

Autoradiography↗