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Biomedical subjects

A L Harris

Publications and source records attributed to A L Harris.

At least 307 records · Page 17Linked to original sources
PubMed1995

Importance of P450 reductase activity in determining sensitivity of breast tumour cells to the bioreductive drug, tirapazamine (SR 4233).

P450 reductase (NADPH:cytochrome P450 reductase, EC 1.6.2.4) is known to be important in the reductive activation of the benzotriazene-di-N-oxide tirapazamine (SR 4233). Using a panel of six human breast adenocarcinoma cell lines we have examined the relationship between P450 reductase activity and sensitivity to tirapazamine. The toxicity of tirapazamine was found to correlate strongly with P450 reductase activity following an acute (3 h) exposure under hypoxic conditions, the drug being most toxic in the cell lines with the highest P450 reductase activity. A similar correlation was also observed following a chronic (96 h) exposure to the drug in air but not following acute (3 h) exposure in air. We have also determined the ability of lysates prepared from the cell lines to metabolise tirapazamine to its two-electron reduced product, SR 4317, under hypoxic conditions using NADPH as an electron donor. The rate of SR 4317 formation was found to correlate both with P450 reductase activity and with sensitivity to tirapazamine, the highest rates of SR 4317 formation being associated with the highest levels of P450 reductase activity and the greatest sensitivity to the drug. These findings indicate a major role for P450 reductase in determining the hypoxic toxicity of tirapazamine in breast tumour cell lines.

Antineoplastic Agents↗
PubMed1995

Relationship between expression of topoisomerase II isoforms and intrinsic sensitivity to topoisomerase II inhibitors in breast cancer cell lines.

Topoisomerase II is a key target for many anti-cancer drugs used to treat breast cancer. In human cells there are two closely related, but differentially expressed, topoisomerase II isoforms, designated topoisomerase II alpha and beta. Here, we report the production of a new polyclonal antibody raised against a fragment of the C-terminal domain of the 180 kDa form of topoisomerase II (the beta isoform), which does not cross-react with the 170 kDa form (the alpha isoform). Using this antibody, together with a polyclonal antibody specific for the 170 kDa isoform of topoisomerase II, we have examined the relationship between the sensitivity of a panel of human breast cancer cell lines to different classes of topoisomerase II inhibitors and cellular levels of the topoisomerase II alpha and beta proteins. We found that sensitivity to amsacrine showed a correlation with the level of expression of topoisomerase II alpha protein, and that sensitivity to etoposide showed a similar correlation with the level of expression of topoisomerase II beta protein. There was also a relationship between sensitivity of these cell lines to mitoxantrone and the cellular level of both isoforms of topoisomerase II. No relationship was found between the level of mRNA for topoisomerase II alpha or beta, and either sensitivity of breast cancer cell lines to topoisomerase II inhibitors or the level of topoisomerase II protein expression.

Antibody Specificity↗
PubMed1995

Topoisomerases II alpha and beta as therapy targets in breast cancer.

Topoisomerase II enzymes play an essential role in human DNA metabolism. They are also recognized as primary targets of a number of anti-cancer drugs used in the treatment of breast cancer, which remains a leading cause of cancer-related death in women. While topoisomerase inhibitors have produced significant response rates in this disease, their use has been limited both by toxicity and by the development of resistance. In this article we review the extensive work which has not only increased our understanding of the biochemistry and molecular biology of type II topoisomerases but also enabled more rational drug design. Such knowledge should translate into increased clinical efficacy in the treatment of breast cancer and other malignancies.

Antineoplastic Agents↗
PubMed1995

Mutations in the p53 gene are not limited to classic 'hot spots' and are not predictive of p53 protein expression in high-grade non-Hodgkin's lymphoma.

We have investigated the relationship between the p53 genotype and phenotype in a series of 22 high-grade non-Hodgkin's lymphomas (NHLs) in which we sequenced the p53 gene open reading frame (exons 2-11). Immunostaining for p53 was already available for these cases. Mutations were found in 10/22 cases (45%) and 3/10 were in exons 4 or 10 outside the classic 'hot spot' regions (exons 5-8). Comparison with immunostaining indicated that, besides cases with the 'expected' patterns (in which gene mutation and protein detection were either both present or both absent) there were also cases in which p53 protein was detected in the absence of any mutation and those with a mutant gene in which the protein was undetectable. These data show that: (1) in high-grade NHLs mutations frequently occur outside the classic hot spot regions and (2) staining for p53 is not predictive of the status of the gene, i.e. whether or not a mutation is present. Therefore in order to document p53 involvement in lymphoid tumours it is necessary both to sequence at least the whole translated open reading frame of the gene and to show evidence of protein expression by immunostaining.

Amino Acid Sequence↗
PubMed1995

Urinary basic fibroblast growth factor in patients with bladder cancer and benign prostatic hypertrophy.

OBJECTIVE: To investigate the role of urinary measurements of an angiogenic factor, basic fibroblast growth factor (bFGF), in the assessment of patients with bladder cancer. PATIENTS AND METHODS: Urine from 83 patients was assayed using a commercially available ELISA for bFGF. Thirty-eight patients had a bladder tumour and 21 had a history of bladder cancer but no disease at the time of testing. Twenty-four patients acted as controls, 16 of whom were about to undergo transurethral resection of the prostate (TURP) for benign prostatic hypertrophy (BPH) and eight who had no urological disease. RESULTS: Median urinary bFGF was higher in patients with active bladder cancer than in those with a clear cystoscopy (5.20 and 2.13 ng/g creatinine, respectively; P < 0.005). Median urinary bFGF was also elevated in patients about to undergo TURP (4.52 ng/g creatinine). Using a threshold value of 6.0 ng/g creatinine, the sensitivity of the test for detecting cancer was 42% and specificity was 88%. At a threshold value of 4.0 ng/g the sensitivity was 62% and the specificity 70%. CONCLUSION: The relationship between urinary basic FGF and the presence of bladder cancer was significant. The test is not sufficiently sensitive or specific to use as a screening test for bladder cancer but may be very useful in monitoring the effectiveness of systemic therapies in bladder cancer. Elevated levels of bFGF in the urine of patients about to undergo TURP suggests a role for bFGF in the pathogenesis of BPH.

Aged↗
PubMed1995

Selectivity of connexin-specific gap junctions does not correlate with channel conductance.

Connexins form a variety of gap junction channels that vary in their developmental and tissue-specific levels of expression, modulation of gating by transjunctional voltage and posttranslational modification, and unitary channel conductance (gamma j). Despite a 10-fold variation in gamma j, whether connexin-specific channels possess distinct ionic and molecular permeabilities is presently unknown. A major assumption of the conventional model for a gap junction channel pore is that gamma j is determined primarily by pore diameter. Hence, molecular size permeability limits should increase and ionic selectivity should decrease with increasing channel gamma j (and pore diameter). Equimolar ion substitution of 120 mmol/L KCl for potassium glutamate was used to determine the unitary conductance ratios for rat connexin40 and connexin43, chicken connexin43 and connexin45, and human connexin37 channels functionally expressed in communication-deficient mouse neuroblastoma (N2A) cells. Comparison of experimental and predicted conductance ratios based on the aqueous mobilities of all ions according to the Goldman-Hodgkin-Katz current equation was used to determine relative anion-to-cation permeability ratios. Direct correlation of junctional conductance with dye transfer of two fluorescein-derivatives (2 mmol/L 6-carboxyfluorescein or 2',7'-dichlorofluorescein) was also performed. Both approaches revealed a range of selectivities and permeabilities for all five different connexins that was independent of channel conductance. These results are not consistent with the conventional simple aqueous pore model of a gap junction channel and suggest a new model for connexin channel conductance and permselectivity based on electrostatic interactions. Divergent conductance and permeability properties are features of other classes of ion channels (eg, Na+ and K+ channels), implying similar mechanisms for selectivity.

Animals↗
PubMed1995

Advanced breast cancer: a phase II trial with gemcitabine.

PURPOSE: In this phase II study, the efficacy and tolerability of gemcitabine were studied in 44 patients with locally advanced or metastatic breast cancer. PATIENTS AND METHODS: Of 40 patients assessable for response, 14 were chemotherapy-naive, seven had received adjuvant chemotherapy, and 19 had received one prior chemotherapy regimen for metastatic disease. Gemcitabine was administered as a 30-minute intravenous infusion once a week for 3 weeks followed by a 1-week rest every 4 weeks. The mean number of completed cycles administered was 2.7 and the mean dosage delivered was 725 mg/m2 per injection. Eighty-one percent of doses were delivered as scheduled. RESULTS: There were three complete responses and seven partial responses, for an overall response rate of 25.0% (95% confidence interval [CI], 12.7% to 41.2%). Four patients were not assessable for efficacy (one had insufficient therapy, two had no bidimensionally measurable disease, and one had neither). All responses were independently validated by an external oncology review board. Responses were observed early in treatment, with a median time to response of 1.9 months. The median survival duration for all 40 assessable patients was 11.5 months. Hematologic toxicity was generally mild, with World Health Organization (WHO) grade 3 and 4 leukopenia occurring in 6.8% and 2.3% of patients and neutropenia in 23.3% and 7.0%, of patients, respectively. The only other grade 4 toxicities were infection and nausea and vomiting in one patient each. One patient was withdrawn due to shortness of breath, possibly drug-related. Flu-like symptoms, which were mild, transient, and treatable with acetominophen, were reported in 6.8% of patients. Only one patient developed alopecia of severity greater than WHO grade 2. CONCLUSION: In view of the single-agent activity seen in advanced breast cancer, modest toxicity profile, and novel mechanism of action, gemcitabine deserves evaluation in breast cancer and is an ideal candidate for combination therapy.

Adult↗
PubMed1995

Clinical importance of the determination of tumor angiogenesis in breast carcinoma: much more than a new prognostic tool.

PURPOSE: To review prognostic and therapeutic applications of angiogenesis research in breast carcinoma. METHODS: We reviewed the (1) biologic role of angiogenesis, particularly in transformation, progression, and metastasis of breast cancer; (2) methods to detect angiogenic activity in human pathology; (3) clinical studies relating clinical outcome of patients with breast cancer to the assessment of angiogenesis; (4) predictive value of angiogenesis for response to anticancer therapies; and (5) pharmacologic characteristics of current antiangiogenic drugs. RESULTS: There is mounting evidence that angiogenesis plays a relevant role in the biologic aggressiveness of breast cancer. Using either immunohistochemical or biochemical methods, several studies have shown a worse prognosis for those patients with tumors with high angiogenic activity. In some studies angiogenesis has an independent prognostic value. The most promising angiogenic inhibitors are under early-phase clinical evaluation in patients with tumors resistant to conventional therapies. Novel therapeutic strategies for breast cancer patients are presented and discussed. CONCLUSION: The majority of the retrospective studies show that angiogenesis is an important new prognostic indicator in early-stage breast carcinoma. This marker should be evaluated in prospective controlled clinical trials to demonstrate whether adjuvant therapies may improve the prognosis of those patients at high risk, eg, those with highly vascularized tumors. Since invasive breast carcinoma has a well defined stromal vascular component and produces angiogenic factors, it seems reasonable to postulate that this tumor may be one of the most responsive to angiogenesis inhibitors given alone or in combination with conventional anticancer treatments.

Breast Neoplasms↗
PubMed1995

Inactivation of Cdc2 increases the level of apoptosis induced by DNA damage.

A number of lines of evidence have suggested a possible involvement of the mitosis-promoting protein kinase Cdc2 in the process of apoptotic cell death, and one recent study concluded that premature activation of Cdc2 is required for apoptosis. Here we have used a temperature-sensitive murine Cdc2 mutant cell line and Cdc2 inhibitor compounds to study the effect of inhibition of this protein kinase on apoptosis induced by DNA-damaging drugs. Inhibition of Cdc2 activity before or during exposure to DNA strand break-inducing drugs had the effect of increasing the level of subsequent apoptosis, as assessed by electron microscopy and flow cytometry. We conclude that, far from being required for cell death, a form of mammalian Cdc2 suppresses apoptosis induced by DNA damage. This form of Cdc2 appears to be active in G2-arrested cells and is therefore presumably distinct from the mitosis-promoting Cdc2-cyclin B heterodimer.

Animals↗
PubMed1995

An immunohistochemical and prognostic evaluation of cathepsin D expression in 105 bladder carcinomas.

Cathepsin D is a widely expressed aspartyl lysosomal protease. Clinical studies in several tumor types have shown a strong correlation between cathepsin D expression and tumor progression. In breast carcinoma, its expression is an independent prognostic factor associated with an increased risk of death. However, there have been no studies evaluating cathepsin D in bladder tumors. Therefore, the aim of this study was to determine the pattern of expression of cathepsin D in a large series of bladder carcinomas and assess its role as a prognostic factor against established variables. The tumors from 105 patients (median age 73) (median follow-up 26 months) with transitional cell carcinoma of bladder were examined. Forty-nine patients had superficial tumors (16 pTa; 33 pT1), 56 had invasive tumors (14 pT2; 42 pT3); there were 35 grade 1/2 tumors and 70 grade 3 tumors. These were stained by a standard immunohistochemical technique with an anti-cathepsin D monoclonal antibody. All 4 normal bladder specimens were positive for cathepsin D. Fifty-four tumors (51%) were positive for cathepsin D and 51 (49%) were negative. Chi square analysis showed a significant positive relationship between negative cathepsin D expression and stage (p < 0.0005), grade (p < 0.0001) and tumor morphology (p = 0.001). There was no relationship between cathepsin D expression and tumor ploidy (p > 0.1) or patient age (p = 0.09). Univariate analysis of disease-free and overall survival showed that negative cathepsin D expression (p = 0.01 and p = 0.0003 respectively), stage (p = 0.004 and p < 0.005 respectively) and grade (p = 0.02 and p = 0.0007 respectively) were associated with significantly worse prognosis. However, in a multivariate analysis of age, stage, grade and cathepsin D expression, only stage remained significant for overall survival (p < 0.005). The observed result for cathepsin D in the univariate analysis is probably due to its strong association with grade and stage. Nevertheless, cathepsin D status was able to provide additional prognostic information for overall survival in invasive tumors when stratifying for grade (p = 0.047), which suggests that it might provide additional prognostic data within particular tumor stages.

Adult↗
PubMed1995

Cyclic AMP-dependent protein kinase type I is involved in hypersensitivity of human breast cells to topoisomerase II inhibitors.

Topoisomerase II (Topo II) is an essential enzyme that catalyzes the breakage of double-strand DNA and is the target of several effective anticancer drugs, including the epipodophyllotoxins. The regulatory subunits of the cyclic AMP-dependent protein kinase are differentially expressed in normal and cancer cells. The RIalpha subunit is overexpressed in cells transformed by transforming growth factor-alpha (TGF-alpha) or Ha-ras oncogene. It has been shown that murine cells transformed by Ha-ras become hypersensitive to Topo II-targeting anticancer drugs. In this report we have tested whether any correlation exists between the expression of RIalpha protein and cellular sensitivity of Topo II-targeting drugs. Normal human breast MCF-10A cells and their derivatives overexpressing TGF-alpha, Ha-ras, or the different protein kinase subunits were treated with either Topo II inhibitors, such as etoposide, teniposide, or amsacrine, or with drugs which act independently of Topo II, such as bleomycin. Here we show that MCF-10A TGF-alpha and MCF-10A Ha-ras cells overexpress the RIalpha protein and become hypersensitive to epypodophyllotoxins and amsacrine but not to bleomycin. Direct introduction of the RIalpha gene into MCF-10A induces hypersensitivity to Topo II inhibitor drugs. In contrast, the overexpression of the other protein kinase subunits, RIIbeta or Calpha, does not modify the drug sensitivity of MCF-10A cells. No differences in the mRNA/protein content or in the activity of Topo II were found between hypersensitive cells and parental MCF-10A cells, suggesting that RIalpha may influence drug sensitivity via modulation of events downstream of the Topo II-DNA cleavable complex.

Amsacrine↗
PubMed1995

Wnt5a cloning, expression, and up-regulation in human primary breast cancers.

Wnt genes are involved in mouse mammary cancer, but their role in human cancer is unknown. Human Wnt5a was cloned from a placental cDNA library and used to assess expression by ribonuclease protection and in situ hybridization in human breast cell lines and in normal, benign, and malignant breast tissues. Human Wnt5a shows over 99% homology at amino acid level with mouse Wnt5a, and 90% with Xenopus Wnt5a. It was expressed only at low levels in breast cell lines and normal breast tissue. Benign proliferations and invasive cancer respectively showed 10-fold and 4-fold higher Wnt5a than normal breast tissues. The greater up-regulation in benign conditions suggests a role in aberrant differentiation. In situ hybridization localized the signal to the epithelial component. Wnt5a is the first member of the Wnt family to demonstrate overexpression in human breast cancer. It was not associated with factors known to affect breast cancer prognosis such as lymph node status or epidermal growth factor receptor status.

Amino Acid Sequence↗
PubMed1994

A role for the human DNA repair enzyme HAP1 in cellular protection against DNA damaging agents and hypoxic stress.

The HAP1 protein (also known as APE/Ref-1) is a bifunctional human nuclear enzyme required for repair of apurinic/apyrimidinic sites in DNA and reactivation of oxidized proto-oncogene products. To gain insight into the biological roles of HAP1, the effect of expressing antisense HAP1 RNA in HeLa cells was determined. The constructs for antisense RNA expression consisted of either a full-length HAP1 cDNA or a genomic DNA fragment cloned downstream of the CMV promoter in pcDNAneo. Stable HeLa cell transfectants expressing HAP1 antisense RNA were found to express greatly reduced levels of the HAP1 protein compared to equivalent sense orientation and vector-only control transfectants. The antisense HAP1 transfectants exhibited a normal growth rate, cell morphology and plating efficiency, but were hypersensitive to killing by a wide range of DNA damaging agents, including methyl methanesulphonate, hydrogen peroxide, menadione, and paraquat. However, survival after UV irradiation was unchanged. The antisense transfectants were strikingly sensitive to changes in oxygen tension, exhibiting increased killing compared to controls following exposure to both hypoxia (1% oxygen) and hyperoxia (100% oxygen). Consistent with a requirement for HAP1 in protection against hypoxic stress, expression of the HAP1 protein was found to be induced in a time-dependent manner in human cells during growth under 1% oxygen. The possible involvement of a depletion of cellular glutathione being linked to the hypoxic stress-sensitive phenotype of the antisense HAP1 transfectants came from the finding that they also exhibited hypersensitivity to buthionine sulphoximine, an inhibitor of glutathione biosynthesis. We conclude that the HAP1 protein is a key factor in cellular protection against a wide variety of cellular stresses, including DNA damage and a change in oxygen tension.

Buthionine Sulfoximine↗
PubMed1994

Repair of cisplatin-DNA adducts by protein extracts from human ovarian carcinoma.

Human ovarian carcinoma exhibits a spectrum of responses to treatment with cisplatin, ranging from marked sensitivity to relative resistance. Variations in the efficiency with which cells repair cisplatin-DNA adducts may in part determine cisplatin sensitivity. We have investigated the use of a novel cell-free assay of nucleotide excision repair of platinated DNA to assess the DNA repair capacity of direct biopsies of untreated human ovarian carcinoma. In this pilot study, ovarian carcinoma extracts performed in vitro excision repair of platinated DNA. Moreover, tumors from different individuals varied as much as 10-fold in their repair capacity in this assay.

Animals↗
PubMed1994

Association of ovarian malignancy with expression of platelet-derived endothelial cell growth factor.

BACKGROUND: At the present time, the pathogenesis of ovarian cancer remains poorly understood, with invasive diagnosis and ineffective treatment for women with the disease. Despite scientific and medical advances in oncology, the overall 5-year survival rate of 30% for ovarian cancer patients has not changed in 20 years. An understanding of the angiogenic process as it occurs in ovarian cancer would not only increase our knowledge of the pathogenesis of this cancer but also might offer novel opportunities for therapeutic intervention. PURPOSE: Our aim was to study the expression of messenger RNA (mRNA) coding for four putative angiogenic factors in normal ovaries and benign and malignant ovarian tumors: platelet-derived endothelial cell growth factor (thymidine phosphorylase), vascular endothelial growth factor, basic fibroblast growth factor, and transforming growth factor-beta 1. METHODS: Four normal ovaries and 25 tumors (seven benign, one of borderline malignancy, and 17 malignant) were collected from 29 patients during elective oophorectomy. The site of sampling (areas of high-velocity blood flow) was directed by transvaginal color Doppler imaging performed within 24 hours of the surgery. Increased blood flow within the tissues was demonstrated by the presence of color (i.e., the velocity was > 7 cm/s) and, together with a pulsatile index of less than 1.0, constituted a positive scanning result. In scan-positive tissues, the area of maximum blood flow was chosen. In scan-negative tissues, a solid area was chosen in complex lesions, or the cyst wall was chosen in simple lesions. Ovarian RNA was subsequently extracted from areas of high-velocity flow (i.e., tissues with a positive scanning result) or from solid areas or septa in tissues with a negative scanning result. A ribonuclease protection assay was used to assess the expression of mRNA coding for the four angiogenic factors. RESULTS: Two normal ovaries (containing a corpus luteum) and one benign and 17 malignant tumors (plus the borderline) gave a positive scanning result. There was a significant difference between the expression of mRNA for platelet-derived endothelial cell growth factor between scan-positive and scan-negative tissues (P < .001) and between benign and malignant tumors (P < .001). CONCLUSIONS: Areas of high blood velocity in ovarian tumors are associated with increased expression of platelet-derived endothelial cell growth factor. IMPLICATIONS: Drugs that affect the angiogenic activity of platelet-derived endothelial cell growth factor offer a potential route for therapeutic intervention.

Autoradiography↗
PubMed1994

Normal human tissues, in addition to some tumors, express multiple different CD44 isoforms.

At least 20 different isoforms of the human CD44 lymphocyte-homing receptor/hyaluronan receptor have been described to date that arise from the differential splicing of up to 10 alternative exons (termed v1-v10) encoding the membrane-proximal extracellular domain. Although numerous analyses at the mRNA level have indicated tissue-specific expression of CD44 variants, few analyses have been performed at the protein level because of limited availability of suitable monoclonal antibodies. Recently, however, exon-specific monoclonal antibodies have been generated using bacterial fusion proteins, and these have been reported to detect high levels of vCD44 containing the v6 exon on human tumors. Together with earlier evidence linking this particular exon with tumor metastasis in the rat, these latter experiments have led to the interpretation that v6 splice variants play a causative role in tumor dissemination. In this paper we describe the use of a new and comprehensive panel of CD44 exon-specific monoclonal antibodies generated against a recombinant CD44(v3-10)-immunoglobulin chimera to study vCD44 expression in a large number of normal and neoplastic tissues. We show that the expression of vCD44 varies greatly among different human tumors and that some express either very low levels of vCD44 or no CD44 at all. Furthermore, we demonstrate that expression is not limited to isoforms containing the v6 exon but includes variants carrying v3, v4/5, and v8/9. Additionally, normal epithelial tissues are shown to express considerable levels of these same vCD44 isoforms. Such results argue against a ubiquitous role for vCD44 isoforms in promoting tumor growth and metastasis.

Antibodies, Monoclonal↗