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Biomedical subjects

A L Harris

Publications and source records attributed to A L Harris.

At least 361 records · Page 20Linked to original sources
PubMed1993

Phosphomonoester is associated with proliferation in human breast cancer: a 31P MRS study.

Phospholipid metabolism of human breast cancer was studied by 31P magnetic resonance spectroscopy (MRS). In vivo localised 31P MR spectra were obtained from the tumour alone using phase modulated rotating frame imaging. For 31 tumours, median (range) phosphomonoester (PME) to ATP ratio was 1.48 (0.57-3.78) and phosphodiester (PDE) to ATP ratio was 1.65 (0.44-3.89). DNA index and S phase fraction (SPF) were measured by flow cytometry of paraffin embedded tissue. Twelve (39%) tumours were diploid and 19 aneuploid. Median (range) SPF for 29 assessable tumours was 5.3% (0.6-28%), with significantly greater median SPF for aneuploid tumours (9.3%) than diploid (3.8%, P = 0.007). There was a significant association between PME/ATP and SPF (P = 0.03) due to a significant correlation for aneuploid tumours (P = 0.01). High resolution 31P MRS of extracts from 18 tumours (including seven studied in vivo) demonstrated that the PME peak consists predominantly of phosphoethanolamine (PE) with a smaller contribution from phosphocholine (PC) (median (range) PE/PC: 3.02 (1.13-5.09)). Changes in PME/ATP were observed for two tumours where tamoxifen stablized disease and may be consistent with the cytostatic effects of this drug.

Adenosine Triphosphate↗
PubMed1993

Differential modulation of doxorubicin toxicity to multidrug and intrinsically drug resistant cell lines by anti-oestrogens and their major metabolites.

The ability of the anti-oestrogens tamoxifen, toremifene and their 4-hydroxy and N-desmethyl metabolites to modify doxorubicin (dox) toxicity to intrinsically resistant and multidrug resistant cell lines was compared, using human breast and lung cancer, and Chinese hamster ovary cell lines. The anti-oestrogens significantly enhanced dox toxicity to multidrug resistant, P-glycoprotein-positive cell lines, but did not affect toxicity to intrinsically resistant, P-glycoprotein-negative cells. Modification was observed at clinically achievable anti-oestrogen concentrations. Toremifene and tamoxifen would therefore appear to be good candidates for in vivo studies as MDR modulating agents in selected patients with P-glycoprotein-positive tumours.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

The relationship of quantitative epidermal growth factor receptor expression in non-small cell lung cancer to long term survival.

Increased expression of epidermal growth factor receptor (EGFr) has been reported in non small cell lung cancers (NSCLC) when compared to normal lung. We have examined post-operative survival in 19 surgically treated patients with NSCLC who had full characterisation of EGFr on primary tumour membrane preparations from resection specimens. There were ten squamous, seven adeno and two large cell carcinomas. The median concentration of high affinity sites was 31 fmol per mg of protein (4-1532) and the median dissociation constant (Kd) of these high affinity sites was 2.3 x 10(-10) per mol (1.2-30 x 10(-10)). Seven patients survived over 5 years. Twelve patients died between 8.5 and 55 months from the time of surgery. When > 5 year survivors were compared to non-survivors there was no difference as regards tumour size or stage, or as regards age or sex. The survivors had a median concentration of high affinity EGFr sites of 16.1 fmol mg-1 protein compared to a median concentration of 68.6 fmol mg-1 protein in the non-survivors (P = 0.01 Wilcoxon test). No long term survivor had > 35 fmol mg-1 protein of receptor. Thus EGFr quantitation may give independent prognostic information in NSCLC and help to select patients for adjuvant therapy after surgery. These results need confirmation in a larger prospective study.

Adenocarcinoma↗
PubMed1993

cDNA transfection followed by the isolation of a MCF-7 breast cell line resistant to tamoxifen in vitro and in vivo.

A tamoxifen resistant cell line (clone 9) has been isolated from the tamoxifen sensitive, hormone responsive MCF-7 breast carcinoma cell line after transfection with mixed cDNA libraries, followed by tamoxifen selection in the presence of oestrogens. Transfection was confirmed by Southern analysis with vector probes. Clone 9 in several-fold more resistant to tamoxifen and other anti-oestrogens than wild type cells when cultured either as a monolayer or as colonies in soft agar but retains oestrogen receptors. Clone 9 was less responsive to 17-beta-oestradiol than were wild type MCF-7. In addition to showing in vitro tamoxifen resistance, clone 9 was also tamoxifen resistant in vivo when xenografted into the nude mouse. Culture medium conditioned by clone 9 cells stimulated quiescent cells of the same clone as well as wild type cells, whereas medium conditioned by wild type MCF-7 was inhibitory to both, suggesting that clone 9 may be secreting an autocrine growth factor. Clone 9 provides a novel model for further investigation of the mechanism of anti-oestrogen resistance that occurs without loss of oestrogen receptors. Preliminary results suggest that an autocrine growth stimulatory mechanism may be one pathway of such resistance.

Animals↗
PubMed1993

Preclinical, phase I and pharmacokinetic studies with the dimethyl phenyltriazene CB10-277.

Decarbazine is an imidazole dimethyltriazene with reproducible activity in patients with metastatic melanoma. CB10-277 is a phenyl dimethyltriazene which, like dacarbazine, requires metabolic activation to its corresponding monomethyl species for antitumour activity. In preclinical models (human melanoma xenografts and transplantable rodent tumours) CB10-277 showed a similar spectrum and level of activity when compared to dacarbazine. Pharmacokinetic studies were performed with CB10-277 in mice treated i.v. at the LD10 (750 mg m-2) and plasma analysed by HPLC. The parent drug area under the plasma concentration vs time curve (AUC) was 142 mM x minutes. Drug metabolism occurred as evidenced by the HPLC identification of the monomethyl species (AUC = 8 mM x minutes) as well as other metabolites. A Phase I trial using a short infusion with doses repeated every 21 days has been performed. Thirty-six patients received 80 courses over a dose range of 80-6,000 mg m-2. The dose limiting toxicity was nausea and vomiting which occurred in 80% of the evaluable courses > or = 900 mg m-2. The only other common side effect was a flushing or warm sensation, which occurred in over 75% of courses at > or = 1,350 mg m-2. There were no hemodynamic consequences. Responses occurred in patients with melanoma (one complete, two partial, one mixed/11), sarcoma (one mixed/6) and carcinoid (one partial/l). Pharmacokinetics were performed in 46 courses. The CB10-277 AUC increased linearly with dose (r = 0.9203, P < 0.001) up to 700 mM x minutes at 6,000 mg m-2). Evidence of CB10-277 metabolism was observed, as in mice, by detection of the monomethyl species and other metabolites. However, the plasma levels of the monomethyl species in patients (1.8 and 3.7 mM x minutes at 6,000 mg m-2) were less than those predicted from studies in mice. Despite this, antitumour activity in dacarbazine sensitive histologies was observed and additional studies with CB10-277 are recommended.

Adult↗
PubMed1993

Hormonal treatment of pancreatic carcinoma: a phase II study of LHRH agonist goserelin plus hydrocortisone.

Eighteen consecutive patients with measurable locally advanced or metastatic pancreatic adenocarcinoma were treated with goserelin (Zoladex) 3.6 mg subcutaneously every 4 weeks. Hydrocortisone 20 milligrams twice daily was commenced with the second injection of goserelin. Objective tumour response was monitored by computerised tomography of the abdomen. There was no objective remission in disease sites. Serial measurements of serum tumour markers showed no reduction in serum CA 19-9 and CA 195 concentrations. The median duration of survival of all cases was 5 months. Administration of goserelin resulted in significant reductions in oestradiol, testosterone, androstenedione in males and reductions in FSH and LH in both males and females. The addition of hydrocortisone resulted in further reductions of androstenedione and testosterone levels in males. Thus goserelin showed no anti-tumour effect, but concentrations required for direct inhibitory effects may be higher than those required to produce effects on hormone suppression.

Adenocarcinoma↗
PubMed1993

The role of DNA topoisomerases II in drug resistance.

The essential nuclear enzyme DNA topoisomerase II is required for the action of a significant number of cytotoxic compounds. Resistance mechanisms identified in cell lines include down-regulation of protein expression, by gene methylation or down-regulation of mRNA, altered drug-DNA-protein interaction or ATP binding, post translational modification of the protein and alteration in expression of the isoenzymes. There is a lack of data relating the findings from these cell lines to observations from clinical practice and the evolution of specific drug resistance in patients. For leukaemias, several studies using different in vitro chemosensitivity assays show a correlation between the clinical response and the in vitro sensitivity (Sargent & Taylor, 1989; Pieters et al, 1989; Larsson et al, 1992; Bosanquet, 1991). From this data, outcome may be related to the mechanism of resistance and allow the development of strategies to overcome them. This includes the use of colony stimulating factors or antimetabolites or the development of new drugs to utilize topoisomerases as their target but which are not transported by P-glycoprotein. Thus an understanding of these mechanisms may help in the optimal use of the topoisomerase II inhibitors.

Animals↗
PubMed1993

Immunohistochemical detection of p53 and bcl-2 proteins in non-Hodgkin's lymphoma.

We have investigated the immunohistochemical expression of p53 protein in 96 cases of non-Hodgkin's lymphoma using a panel of five antibodies. Positive neoplastic cells were found in 30 (31.2%) cases, which could be divided into two groups according to their patterns of reactivity with the different antibodies; i.e. those positive with both polyclonal and monoclonal antibodies, and those which were stained only by monoclonal antibodies PAb1801 and/or PAb240. Positivity was nuclear in all but six cases in which cytoplasmic staining was found. In view of the hypothesis recently raised that p53 protein induces apoptosis we have compared our results with parallel staining for bcl-2 protein since bcl-2 is believed to be important, at least in lymphomas, in suppression of apoptosis. Staining for bcl-2 protein was performed on 83 cases and it was shown that p53-positive cases accounted for 10 out of 17 (59%) of the bcl-2-negative lymphomas but only for 15 out of the 66 (23%) bcl-2-positive cases, suggesting a possible relationship between the expression of these two proteins. Thus, our data show that p53 protein is abnormally expressed in a substantial proportion of non-Hodgkin's lymphomas and bears a significant inverse relationship to bcl-2 protein expression. However the molecular basis of this expression remains to be elucidated.

Humans↗
PubMed1993

p53 expression in colorectal adenomas.

To assess the expression of p53 in premalignant lesions, we examined by immunohistochemistry benign colorectal adenomas (n = 72, measuring more than 6 mm and less than 95 mm in diameter) from patients without (group I, n = 23) or with (group II, n = 49) concurrent sporadic colorectal carcinomas. Using a panel of three monoclonal antibodies (PAb 240, PAb 421, PAb 1801) and two polyclonal antibodies (CM1, C19) immunohistological staining was demonstrated in 26% of the cases (19 of 72 adenomas, 7 of 23 from group I and 12 of 49 from group II). In the majority of the cases, p53 positive foci in the adenomas occurred in the most dysplastic areas, although focal positivity was detected in glands that were histologically normal. Expression of p53 protein was also detected in 21 of 30 (70%) colorectal carcinomas of group II. In two cases focal positive staining was observed in the polyps but not in the concurrent carcinomas. Non-neoplastic colonic mucosa and stromal lymphoid cells were negative in all cases examined. Over-expression of p53 in neoplastic tissues detected by immunocytochemistry is generally believed to correlate with the presence of mutation in the gene. This may not be an absolute rule, because in some hemopoietic malignancies, there is evidence that p53 protein may be detectable in the absence of an underlying mutation. These findings therefore represent the highest incidence in colorectal adenomas of abnormalities in the p53 protein expression, probably largely due to underlying mutations. This study also suggests that immunocytochemical demonstration of p53 protein may be a suitable method for the routine detection of subpopulations of cells which, by clonal expansion, could acquire a growth advantage within an adenoma during the neoplastic process.

Adenoma↗
PubMed1993

Topoisomerase II alpha co-amplification with erbB2 in human primary breast cancer and breast cancer cell lines: relationship to m-AMSA and mitoxantrone sensitivity.

Topoisomerase II alpha (topo II alpha) is a key enzyme in DNA replication and a target for many anti-cancer drugs. High levels are associated with sensitivity to topoisomerase II inhibitors. Because its chromosome location is similar to erbB2 (17q21-22), which is frequently amplified in breast cancer, co-amplification of these genes was assessed. In 117 primary breast cancers, 25 were amplified for erbB2. Three of these cases showed co-amplification of topo II alpha. Topo II beta was not amplified. Four human breast cancer cell lines were assessed for erbB2 and topo II alpha co-amplification. They were also analysed for sensitivity to the topoisomerase inhibitors m-AMSA and mitoxantrone. The most sensitive cell line was SKBr3, which was the only one with erbB2 amplification. Topo II alpha was co-amplified to a similar extent as in tumours. This suggests that patients whose tumours show topo II alpha amplification should be assessed specifically for therapy with topoisomerase inhibitors.

Adult↗
PubMed1992

Isolation of cDNA clones encoding the beta isozyme of human DNA topoisomerase II and localisation of the gene to chromosome 3p24.

Topoisomerases catalyse the interconversion of topological isomers of DNA and have key roles in nucleic acid metabolism. Human cells express two distinct type II topoisomerase isozymes, designated topoisomerase II alpha (170 kDa form) and topoisomerase II beta (180 kDa form). We have isolated cDNA clones encoding the beta isozyme from a human B-cell library. The proposed coding region for the topoisomerase II beta protein is 4,863 nucleotides long and would encode a polypeptide with a calculated M(r) of 182,705. The predicted topoisomerase II beta protein sequence shows striking similarity (72% identical residues) to that of the human alpha isozyme, and homology to topoisomerase II proteins from Drosophila, yeast and bacteria. Regions of greatest amino acid sequence divergence lie at the extreme N-terminus and over a C-terminal domain comprising approximately 25% of the total protein. We have quantified the level of topoisomerase II beta mRNA in a panel of human tumour cell lines of different origin using an RNase protection assay, and compared the level to that of topoisomerase II alpha mRNA. Topoisomerase II beta mRNA was expressed in haemopoietic, epithelial and fibroblast cell lines, although to different extents, with U937 cells (promonocytic leukaemia) showing a particularly high level. There was no obvious relationship in terms of level of expression between the topoisomerase II alpha and beta genes. We have localised the gene encoding topoisomerase II beta protein to chromosome 3p24 in the human genome.

Amino Acid Sequence↗
PubMed1992

Angiogenesis, assessed by platelet/endothelial cell adhesion molecule antibodies, as indicator of node metastases and survival in breast cancer.

Animal models suggest a role for new vessel formation (angiogenesis) in tumours with metastatic potential, and there is some evidence that this is true for human tumours. What is needed is a sensitive and specific label for endothelial cells, and one candidate would be a monoclonal antibody to platelet/endothelial cell adhesion molecule (PECAM). We have counted microvessels in 103 primary breast cancers using the JC70 antibody to PECAM (or CD31). We compared our findings with various pathological indicators (lymph node status and tumour grade, size, and type and markers (oestrogen receptor, and c-erbB-2 expression and detection of mutant p53). Tumours showed significantly higher vascularisation than normal breast tissue and the number of blood vessels/mm2 was significantly associated with node metastasis. Only 2 out of 50 tumours with 99 vessel/mm2 or less were node positive whereas 31 out of 39 tumours with counts above 140/mm2 were positive (p < 0.0001). Tumour size and grade also correlated with node metastasis and vascularisation also increased with the size of the primary and with poor differentiation. However, within each subgroup of size or differentiation tumours without node involvement had much lower vascular counts, and multivariate analysis showed that vascular count alone explains the association of size and grade with node metastasis. Other markers, conventional or novel, did not correlate with vascularisation. Even with the short follow-up in this series, vascular counts correlated with early death. These results suggest that angiogenesis is closely linked to metastasis, that it is acquired at a critical density of vessels, and that this process occurs as tumours enlarge or become more poorly differentiated. Counting of newly formed microvessels stained with endothelium-specific antibodies may prove to be a useful tool in the early detection of metastatic potential and in the selection of patients for whom anti-angiogenesis drugs might be beneficial.

Antigens, Differentiation, Myelomonocytic↗
PubMed1992

Cloning and characterization of the 5'-flanking region of the human topoisomerase II alpha gene.

Topoisomerases are essential enzymes for DNA metabolism in prokaryotes and eukaryotes. In human cells, DNA topoisomerase II enzyme activity can be modulated by both viral transformation and changes in proliferation status. To identify elements important for regulation of topoisomerase II alpha gene expression, genomic DNA clones covering the 5'-end of the gene were isolated. The intron/exon structure of a 2.5-kilobase region encompassing the translation start site was determined. Transcription was found to initiate at multiple sites clustered around 90 base pairs 5' to the ATG initiation codon. Transient expression of chimeric topoisomerase II-reporter gene constructs in HeLa cells revealed that the 5'-flanking region exhibited promoter activity. The region -90 to -1 upstream of the major transcription start site was shown by deletion analysis to include a promoter. This minimal promoter lacks a TATA box, is moderately GC-rich, and contains a high frequency of CpG dinucleotides; characteristic of a "housekeeping" gene promoter. Maximal promoter activity was observed using a fragment extending to position -562. Putative regulatory elements are contained within and immediately upstream of the minimal promoter region. The regulatory region of the topoisomerase II alpha gene identified here is similar in basic structure to those of the human thymidine kinase and DNA polymerase alpha genes, which are also controlled by proliferation-specific factors.

Base Sequence↗
PubMed1992

Loss of HLA class-I alleles, heavy chains and beta 2-microglobulin in colorectal cancer.

Using immunohistochemical methods, we have analysed colorectal biopsies of normal mucosa, metaplastic polyps (5 cases), adenomas (15 cases) and adenocarcinomas (70 cases) with 13 monoclonal antibodies (MAbs) to allelic products of the HLA-A, B, C loci. Nine of the 70 carcinomas showed total loss of HLA Class-I molecules due to an underlying defect regarding not only the expression of beta 2-microglobulin (beta 2-m), but also the heavy chains of HLA A, B and C loci, or both. Much commoner was a loss of one or more Class-I alleles as follows: A1/Aw36 (completely lost in 4 of 29 cases and focally lost in another 2), A2 (in 1 of 37 cases), A3 (in 2 of 14 cases), A1 1/28/31/33 (in 3 of 11 cases), B7 (in 3 of 13 and focally in 1 other case), B17 (in 1 case), Bw4 (in 8 of 45 and focally in another 6), Bw6 (in 9 of 62 and focally in another 3). Focal selective loss (Bw6 and a combined A1-Bw6), was observed in 2 adenomas. Normal colonic mucosa, as well as stromal and lymphoid cells present between the neoplastic glands, were studied in each case as a control. A particular allele was only considered to be lost by the malignant cells if it was still expressed on these adjacent tissues.

Alleles↗
PubMed1992

Vascular protein kinase C in Wistar-Kyoto and spontaneously hypertensive rats.

Phorbol esters which activate protein kinase C (PKC) produced concentration-related force development in aorta from spontaneously hypertensive rat (SHR) and Wistar-Kyoto rat (WKY); all were 2-7 x more potent in SHR. However, total PKC activity in aortas, as well as carotid, caudal and renal arteries, was not different, when SHR was compared with WKY. Binding of phorbol dibutyrate to particulate aortic PKC was similar in SHR and WKY (same apparent Kd and Bmax values), as was potency for displacement of phorbol dibutyrate by phorbol myristate acetate. Furthermore, there was no difference in potency with staurosporine, H-7, and calmidazolium in inhibiting SHR and WKY aortic PKC. These data demonstrate enhanced contractile sensitivity to PKC-activating phorbol esters in SHR aortic smooth muscle that is not related to activity, phorbol ester binding, or sensitivity to inhibitors when SHR PKC is compared with WKY PKC. Thus, signal transduction events distal to PKC activation may be responsible for enhanced vascular contractile sensitivity to phorbol esters in SHR.

Animals↗
PubMed1992

Reduced topoisomerase II and elevated alpha class glutathione S-transferase expression in a multidrug resistant CHO cell line highly cross-resistant to mitomycin C.

We have isolated a multidrug-resistant derivative of Chinese hamster ovary CHO-K1 cells by exposure to progressively increasing concentrations of Adriamycin. This cell line, designated CHO-Adrr, was 27-fold more resistant than the parental line to Adriamycin and showed similar degrees of cross-resistance to several other topoisomerase II (topo II) inhibitors, including mitoxantrone, daunomycin and etoposide. CHO-Adrr cells showed a lower (4-fold) level of cross-resistance to vincristine and colchicine, drugs associated with the multidrug-resistant phenotype. While CHO-Adrr cells showed no enhanced resistance to several mono- and bi-functional alkylating agents or to UV and ionizing radiation, they were greater than 80-fold resistant to mitomycin C (MMC). There was a 5-fold decreased level of daunomycin accumulation in CHO-Adrr cells compared to CHO-K1 cells and this was associated with increased drug efflux. The resistant cells had amplified multidrug resistance gene (mdr) sequences and overexpressed (mdr) mRNA. Verapamil was able to completely reverse Adriamycin resistance but reversal of MMC resistance was only partial, with residual 23-fold resistance. CHO-Adrr cells expressed a 4-fold reduced level of topo II protein but overexpressed an alpha class (basic) glutathione S-transferase (GST). Analysis of cell hybrids showed that while the level of resistance to Adriamycin dropped by a factor of 3 in CHO-K1/CHO-Adrr hybrids compared to CHO-Adrr/CHO-Adrr hybrids, resistance to MMC dropped 10-fold. Thus, CHO-Adrr cells appear to exhibit simultaneously several different drug resistance mechanisms including MDR and GST overexpression, and topo II reduction.

Amsacrine↗
PubMed1992

Inhibition of colon and breast carcinoma cell growth by interleukin-4.

Within human carcinomas, there is often an infiltration of lymphocytes and other cells of the immune system. A variety of cytokines are produced by such cells that could have a paracrine influence on the growth of tumor epithelium. The effect of one of these cytokines, interleukin-4 (IL-4), on human breast and colon cancer cell lines was therefore examined. IL-4 inhibited the growth of human colon (HT 29) and breast [MCF-7 wild type (MCF-7 WT), MCF-7 Adriamycin-resistant (MCF-7r), MDA-MB-231, and MDA-MB-468] carcinoma cells in culture. Competitive binding of 125I-IL-4 demonstrated the presence of 2000 high affinity IL-4-binding sites on HT 29 cells. The Kd for specific binding of 125I-IL-4 to HT 29 cells was 77 pM. Further studies were conducted on the estrogen-dependent MCF-7 WT and estrogen-independent MDA-MB-231 breast carcinoma lines. Concentrations of IL-4 of 10-100 nM were required to significantly inhibit growth of these carcinoma cell lines; e.g., with MCF-7 WT cells, half-maximal inhibition of growth occurred at 20 nM IL-4. Specific binding of 125I-IL-4 was detected to MCF-7 WT and MDA-MB-231 cells, but the low level of binding precluded Scatchard analysis. IL-4 inhibited 90% of the 17 beta-estradiol-stimulated growth of MCF-7 WT cells in a dose-dependent manner but without a change in estrogen receptor expression. Inhibition of growth by IL-4 was less in the absence of estrogens. Combined treatment with IL-4 and other known inhibitors of breast carcinoma cell growth [transforming growth factor-beta 1 (TGF-beta 1) and the antiestrogen tamoxifen] showed additive inhibition. The hormone-independent cell lines MCF-7r and MDA-MB-231 were additively inhibited by IL-4 and TGF-beta 1. This was not the case with MDA-MB-468 cells in which inhibition by IL-4 and TGF-beta 1 was of similar magnitude but no significantly greater effect was observed on combined treatment. No secretion of IL-4 was detected from these cell lines either basally or on treatment with TGF-beta 1 or tamoxifen, and we conclude that IL-4 is a nonautocrine inhibitor of breast carcinoma cell growth.

Breast Neoplasms↗