Discrepancies in central review re-testing of patients with ER-positive and HER2-negative breast cancer in the OPTIMA prelim randomised clinical trial.

BACKGROUND: There is limited data on results of central re-testing of samples from patients with invasive breast cancer categorised in their local hospital laboratories as oestrogen receptor (ER) positive and human epidermal growth factor receptor homologue 2 (HER2) negative. METHODS: The Optimal Personalised Treatment of early breast cancer usIng Multiparameter Analysis preliminary study (OPTIMA prelim) was the feasibility phase of a randomised controlled trial to validate the use of multiparameter assay-directed chemotherapy decisions in the UK National Health Service (NHS). Eligibility criteria included ER positivity and HER2 negativity. Central re-testing of receptor status was mandatory. RESULTS: Of the 431 patients tested centrally, discrepant results between central and local laboratory results were identified in only 19 (4.4%; 95% confidence interval 2.5-6.3%) patients (with 21 tumours). On central review, seven patients had cancers that were ER-negative (1.6%) and 13 (3.0%) patients with 15 tumours had HER2-positive disease, including one tumour discrepant for both biomarkers. CONCLUSIONS: Central re-testing of receptor status of invasive breast cancers in the UK NHS setting shows a high level of reproducibility in categorising tumours as ER-positive and HER2-negative, and raises questions regarding the cost effectiveness and clinical value of central re-testing in this sub-group of breast cancers in this setting.

Comparison of three mathematical models for predicting the risk of additional axillary nodal metastases after positive sentinel lymph node biopsy in early breast cancer.

BACKGROUND: Women with breast cancer and a positive axillary sentinel lymph node (SLN) are recommended to undergo complete axillary lymph node dissection; however, further nodal disease is not always present. Mathematical models have been constructed to determine the risk of metastatic disease; three of these were evaluated independently. METHODS: Data from 108 women with breast cancer who had a positive SLN biopsy and completion axillary lymph node dissection were used. Measurements of additional parameters over those usually determined (such as size of SLN metastasis) were assessed under the supervision of two pathologists. These data were used to determine the predicted risk of non-SLN metastases using three mathematical models (from Memorial Sloan-Kettering Cancer Center (MSKCC), Cambridge University and Stanford University) and a comparison made with the observed findings. Analyses were made using the area under the receiver operating characteristic (ROC) curve (AUC). RESULTS: Some 53 (49.1 per cent) of 108 patients had a positive non-sentinel axillary lymph node metastasis. The AUC values were 0.63, 0.72 and 0.67 for the MSKCC, Cambridge and Stanford nomograms respectively. CONCLUSION: This independent comparison found no significant difference between the models, although the Cambridge model had the advantage of requiring fewer measurements with a more accurate predictive performance.

Cell-cycle-phase progression analysis identifies unique phenotypes of major prognostic and predictive significance in breast cancer.

Multiparameter analysis of core regulatory proteins involved in G1-S and G2-M cell-cycle transitions provides a powerful biomarker readout for assessment of the cell-cycle state. We have applied this algorithm to breast cancer to investigate how the cell cycle impacts on disease progression. Protein expression profiles of key constituents of the DNA replication licensing pathway (Mcm2, geminin) and mitotic machinery (Plk1, Aurora A and the Aurora substrate histone H3S10ph) were generated for a cohort of 182 patients and linked to clinicopathological parameters. Arrested differentiation and genomic instability were associated with an increased engagement of cells into the cell division cycle (P<0.0001). Three unique cell-cycle phenotypes were identified: (1) well-differentiated tumours composed predominantly of Mcm2-negative cells, indicative of an out-of-cycle state (18% of cases); (2) high Mcm2-expressing tumours but with low geminin, Aurora A, Plk1 and H3S10ph levels (S-G2-M progression markers), indicative of a G1-delayed/arrested state (24% cases); and (3) high Mcm2-expressing tumours and also expressing high levels of the S-G2-M progression markers, indicative of accelerated cell-cycle progression (58% of cases). The active cell-cycle progression phenotype had a higher risk of relapse when compared with out-of-cycle and G1-delayed/arrested phenotypes (HR=3.90 (1.81-8.40, P<0.001)), and was associated with Her-2 and triple negative subtypes (P<0.001). It is of note that high-grade tumours with the G1-delayed/arrested phenotype showed an identical low risk of relapse compared with well-differentiated out-of-cycle tumours (HR=1.00 (0.22-4.46), P=0.99). Our biomarker algorithm provides novel insights into the cell-cycle state of dynamic tumour cell populations in vivo. This information is of major prognostic significance and may impact on individualised therapeutic decisions. Patients with an accelerated phenotype are more likely to derive benefit from S- and M-phase-directed chemotherapeutic agents.

Binding of the transcription factor EBP-80 mediates the methylation response of an intracisternal A-particle long terminal repeat promoter.

Intracisternal A-particle (IAP) expression in mouse cells has been correlated with hypomethylation of HhaI and HpaII sites in proviral long terminal repeats (LTRs). In a previous study, in vitro methylation of three HhaI sites in the U3 region of the LTR from the cloned genomic IAP element, MIA14, was shown to inhibit promoter activity in vivo. In this study, we found by site-directed mutagenesis that the two more downstream HhaI sites within this LTR were responsible for the methylation effects on promoter activity in vivo; methylation of the other (5') HhaI site, which lies within a putative SP1 binding domain, did not affect promoter activity. Methylation of the HhaI sites also inhibited promoter activity of the LTR in a cell-free transcription system. Exonuclease III footprinting demonstrated methylation-induced changes in protein binding over the region encompassing the downstream HhaI site, designated the Enh2 domain. The protein that interacts specifically with this domain, EBP-80, was characterized in a previous study (M. Falzon and E. L. Kuff, J. Biol. Chem. 264:21915-21922, 1989). We show here that the presence of methylcytosine in the HhaI site within the Enh2 domain inhibited binding of EBP-80 in vitro. The methylated MIA14 LTR construct was much less responsive to added EBP-80 in an in vitro transcription system than was the unmethylated construct. These data suggest that CpG methylation within the Enh2 domain may exert its effect on transcription in vivo by altering the interaction between EBP-80 and its cognate DNA sequence.

Metabolic activation and cytotoxicity of 4-ipomeanol in human non-small cell lung cancer lines.

In the normal lungs of many animal species, 4-ipomeanol is transformed to a highly reactive metabolite preferentially in pulmonary bronchiolar Clara cells and to a lesser extent in alveolar type II cells, potentially leading to damage or destruction of these cell types. Since Clara cells and type II cells are suspected sites of origin of certain "non-small cell" lung cancers, the metabolic activation of 4-ipomeanol (measured by the metabolism-dependent covalent binding of 4-ipomeanol to cellular macromolecules) was compared in two human non-small cell carcinoma derived cell lines (NCI-H322 and NCI-H358) and two human small cell carcinoma derived cell lines (NCI-H128 and NCI-H69). Metabolic activation of 4-ipomeanol was evident in the non-small cell lines; the production of covalently bound metabolite was somewhat greater in NCI-H322 (morphology related to Clara cells) compared to NCI-H358 (morphology related to alveolar type II cells), but was entirely undetectable in the small cell lines. The activation pathway was concentration (4-ipomeanol) and time dependent and followed Michaelis-Menten kinetics. Metabolism to the reactive intermediate required oxygen and was strongly inhibited by carbon monoxide. Covalent binding was enhanced in the non-small cell lines by prior incubation with beta-naphthoflavone and by supplementation of the incubate with exogenous reduced nicotinamide adenine dinucleotide phosphate. 4-Ipomeanol was more cytotoxic to the non-small cell lines than to the small cell lines under the in vitro growth conditions used. These studies indicate that certain human non-small cell lung cancers have metabolic characteristics of normal bronchiolar Clara cells and alveolar type II cells; these results would therefore be consistent with an origin of these tumors from Clara cells or type II cells, respectively. The present studies indicate that the further preclinical testing and development of 4-ipomeanol is warranted, with a view toward possible clinical evaluation against human lung cancers.

Metabolic activation and biological effects of nitrosamines in the mammalian lung.

Nitrosamines and their precursors are among the most common contaminants of our environment, and many of them are highly carcinogenic. Nitrosamines are believed to require metabolic activation in the host organism, and many of them demonstrate a pronounced organ and cell type specificity. This review summarizes recent in vivo and in vitro experiments which focus on the mechanisms of nitrosamine-induced lung carcinogenesis. Currently available in vivo and in vitro data suggest that nitrosamines may be metabolized by cytochrome P-450, prostaglandin endoperoxide synthetase, or monoamine oxidases. The presence of one or the other of these enzyme systems may be partially responsible for the cell type-specific effects of this class of chemicals. Moreover, evidence in vitro suggests selective uptake of nitrosamines by cell type-specific receptors, a phenomenon which offers a more logical explanation than previously published theories for the selectivity of biological effects exerted by nitrosamines.

DNA sequences in the rat parathyroid hormone-related peptide gene responsible for 1,25-dihydroxyvitamin D3-mediated transcriptional repression.

Expression of the gene encoding PTH-related peptide (PTHrP), a protein that plays a primary role in the development of humoral hypercalcemia of malignancy, is down-regulated at the transcriptional level by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Deletions of the 5'-flanking region of the rat PTHrP gene, when fused to the chloramphenicol acetyl-transferase gene and transfected into ROS 17/2.8 (rat osteosarcoma) cells, showed that the 1,25-(OH)2D3 responsive region is located between -1.05 and -0.71 kb upstream of the transcription start site. Further mapping of this region revealed that a 123-bp fragment is able to confer 1,25-(OH)2D3 responsiveness to a heterologous (SV40) promoter. This region contains two potential vitamin D response elements (VDREs). One of these motifs resembles the negative VDRE (nVDRE) from the PTH gene, which is also down-regulated by vitamin D3. The other element resembles the canonical VDRE (two hexanucleotide motifs separated by three nucleotides), which has been characterized in a number of genes whose expression is modulated by vitamin D3. Electrophoretic mobility shift assays using nuclear extracts from ROS 17/2,8 cells and from vitamin D receptor. (VDR)-enriched COS 1 cells revealed that both elements interact with the VDR. This protein-DNA interaction is disrupted by an anti-VDR antibody. Therefore, modulation of PTHrP gene transcription by 1,25-(OH)2D3 is mediated by the VDR interacting with one or both of the identified motifs in the 5'-flanking sequence of the gene.

Histological classification of the non-Hodgkin's lymphoma.

The non-Hodgkin's lymphomas (NHL) are a diverse group of neoplasms which show subtle histological differences. This has led to difficulties in formulating reproducible classifications of NHL that are both histologically and clinically relevant. In this review we discuss some of the historical aspects of NHL classification leading up to the principal classifications in current use. In comparing these we put forward reasons why we regard the updated Kiel classification as that which is the most soundly based in clinical, histological and biological terms. The Kiel classification, unlike its competitors, lends itself to further updating as new clinicopathological entities become established, without loss of its essentially sound foundations. Thus the classification of T-cell lymphomas can only be regarded as provisional and certain extranodal lymphomas, which are clear clinicopathological entities, await classification. The biological foundation of the Kiel classification should allow further updating to incorporate these entities and the tighter definitions of categories of NHL that will surely result from the application of cytogenetics, molecular biology and studies of lymphocyte homing mechanisms.

Serum stimulation of parathyroid hormone-related peptide gene expression in ROS 17/2.8 osteosarcoma cells through transcriptional and posttranscriptional mechanisms.

The gene encoding PTH-related peptide (PTHrP), a protein that plays a primary role in the development of humoral hypercalcemia of malignancy, is widely expressed in normal and neoplastic tissues. This study demonstrates that expression of the PTHrP gene has features of early response genes, including up-regulation after serum repletion of serum-starved ROS 17/2.8 (rat osteosarcoma) cells. The PTHrP messenger RNA (mRNA) levels were induced within 30 min and peaked at 4 h. Elevated mRNA levels were accompanied by an increase in secreted PTHrP. The serum effects on PTHrP mRNA levels were blocked by actinomycin D, suggesting a requirement for gene transcription. Nuclear run-on assays revealed a 3-fold increase in PTHrP gene transcription 4 h after exposure to serum. Deletions of the 5' flanking sequence of the rat PTHrP gene fused to the chloramphenicol acetyltransferase gene and transfected into ROS 17/2.8 cells showed that the serum-responsive region is located between -1.05 kb and -0.3 kb upstream of the transcription start site. PTHrP mRNA levels were also induced by cycloheximide, another feature common to early response genes. The PTHrP mRNA half-life in serum-starved cells was 56 min. Serum treatment prolonged the half-life 2.7-fold, suggesting serum-induced stabilization of the mRNA. Insulin and epidermal growth factor also induced PTHrP mRNA expression in a time-dependent manner analogous to serum, indicating that the effects of serum may be mediated, at least partially, through these agents. In summary, serum up-regulated PTHrP mRNA expression through both transcriptional and posttranscriptional mechanisms. This rapid stimulation by growth factors suggests that PTHrP may contribute to the early cellular response after growth factor stimulation.

Enhanced growth of MCF-7 breast cancer cells overexpressing parathyroid hormone-related peptide.

PTH-related peptide (PTHrP) is a secreted protein produced by breast cancer cells both in vivo and in vitro. Because of its structural similarity to PTH at the amino terminus, the two proteins interact with a common cell surface receptor, the PTH/PTHrP receptor. When overproduced by tumor cells, PTHrP enters the circulation, giving rise to the common paraneoplastic syndrome of humoral hypercalcemia of malignancy. Although initially discovered in malignancies, PTHrP is now known to be produced by most cells and tissues in the body. It acts as an autocrine and paracrine mediator of cell proliferation and differentiation, effects which are mediated via the PTH/PTHrP receptor. Recent evidence also has shown that, directly after translation, PTHrP is able to enter the nucleus and/or nucleolus and influence cell cycle progression and apoptosis. In this study, we have either overproduced PTHrP or inhibited endogenous PTHrP production in the breast cancer cell line, MCF-7. Overexpression of PTHrP was associated with an increase in mitogenesis, whereas inhibiting endogenous PTHrP production resulted in decreased cell proliferation. The overexpressed peptide targeted to the perinuclear space. In contrast, PTHrP interaction with the cell surface PTH/PTHrP receptor resulted in decreased cell proliferation in the same cell line. This latter effect is dependent on interaction with the receptor, in that exogenously added PTHrP moieties known not to interact with the receptor had no effect on cell growth. Furthermore, neutralization of added peptide with an anti-PTHrP antiserum completely abolished the growth inhibitory effects. In contrast, this antibody has no effect on the increased proliferation rate of the MCF-7 transfectants that overexpress PTHrP, compared with control cells. The net effect of autocrine/paracrine and intracrine effects of PTHrP in MCF-7 cells overproducing the peptide is accelerated cell growth. These findings have critical implications regarding the role of PTHrP in breast cancer, and they suggest that controlling PTHrP production in breast cancer may be useful therapeutically.

The noncalcemic vitamin D analogs EB1089 and 22-oxacalcitriol suppress serum-induced parathyroid hormone-related peptide gene expression in a lung cancer cell line.

PTH-related peptide (PTHrP) mediates the syndrome of humoral hypercalcemia of malignancy, a frequent complication of squamous cell carcinomas of the lung. This study was undertaken to determine whether 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and two nonhypercalcemic analogs, EB1089 and 22-oxa-1,25-(OH)2D3 (22-oxacalcitriol), suppress serum- and epidermal growth factor (EGF)-induced PTHrP gene expression in a human lung squamous cancer cell line, NCI H520. PTHrP expression was up-regulated by serum and EGF in a concentration- and time-dependent manner. Nuclear run-on analysis showed that this induction was mediated via a transcriptional mechanism, and that sequences within promoter 1 were responsible. All three vitamin D3 compounds decreased both basal and serum- and EGF-induced steady state PTHrP messenger RNA and secreted peptide levels. These effects were again mediated via a transcriptional mechanism through sequences within promoter 1. All three vitamin D3 compounds also decreased the proliferation of NCI H520 cells in a concentration- and time-dependent manner. 1,25-(OH)2D3 is hypercalcemic in vivo. However, the noncalcemic analogs EB1089 and 22-oxa-1,25-(OH)2D3 have therapeutic potential, as they suppress not only the basal but also the growth factor-stimulated levels of PTHrP in a cancer cell line associated with hypercalcemia.

Overexpression of parathyroid hormone-related protein enhances apoptosis in the rat intestinal cell line, IEC-6.

We used the rat intestinal cell line, IEC-6, to study potential effects of overexpression of PTH-related protein (PTHrP) on apoptosis. A clonal line of PTHrP-overexpressing cells was established by stably transfecting parental cells with PTHrP complementary DNA in a sense orientation (sense). A similarly transfected line stably, transfected with empty vector, served as control (vector). Immunoreactive PTHrP, measured in culture medium, showed that sense cells secreted approximately 30 times as much PTHrP as did vector control cells. Apoptosis induced by serum withdrawal was evaluated by several methods. DNA laddering was demonstrable in sense-transfected cells as early as 12 h after serum withdrawal but not until later time points in vector-transfected control cells. Flow cytometric analysis of propidium iodide-stained cells showed a greater increase in the sub-G1 (apoptotic) population in sense cells, compared with vector. Fluorescent microscopy with Hoechst 33258 dye showed increased nuclear fragmentation and condensation in sense cells. Studies of apoptotic gene expression by ribonuclease protection assay, and protein by Western blot analysis, showed an enhanced ratio of Bax to Bcl-x(L) in sense cells. Mutation of the PTHrP nuclear localization amino acid sequence negated the ability of PTHrP to enhance apoptosis.

Characterization of human and rat glucagon-like peptide-1 receptors in the neurointermediate lobe: lack of coupling to either stimulation or inhibition of adenylyl cyclase.

Glucagon-like peptide-1 (GLP-1) has been shown to bind to the posterior pituitary in the rat. We examined GLP-1 binding sites in human postmortem and rat pituitaries. Dense [125I]GLP-1 binding was seen in both human and rat posterior pituitary. In rat neurointermediate lobe membranes the binding site showed a Kd of 0.2 +/- 0.01 nM and a binding capacity of 600 +/- 33 fmol/mg protein (n = 3). In human pituitary membranes the binding site showed a Kd of 0.82 +/-0.05 nM and a binding capacity of 680 +/- 93 fmol/mg protein (n = 3). Chemical cross-linking showed a relative mol wt for the receptor-ligand complex of 73,100 +/- 1,400 (n = 3) in man and 59,300 +/- 900 (n = 3) in rat. GLP-1 (1 microM) failed to increase cAMP levels measured in rat neurointermediate lobes, whereas pituitary adenylate cyclase-activating polypeptide (100 nM) increased cAMP from a basal level of 14 +/-1 to 80 +/- 4 pmol/neurointermediate lobe 15 min (n = 5; P < 0.01). GLP-1 (up to 1 microM) did not affect the pituitary adenylate cyclase-activating polypeptide-stimulated cAMP levels. GLP-1 (up to 1 microM) also did not stimulate release of vasopressin or oxytocin from isolated rat neurointermediate lobes. The posterior pituitary shows the highest density of GLP-1-binding sites yet seen, but their function and signal transduction mechanism remain unknown.

Cloning and expression of rat homeo-box-containing sequences.

Six rat homeo-box-containing DNA sequences have been isolated by screening a genomic library with a probe derived from an Antennapedia (Antp) cDNA clone of Drosophila melanogaster. Sequence determination of two of the clones containing the homeo-box regions reveals a 180-nucleotide(nt) domain sharing more than 80% homology at the nucleotide level and more than 90% homology at the amino acid level with the homeo-box from the Antp gene and from homeo-boxes of other metazoan species. Genomic blotting experiments suggest that the two homeo-box-containing DNA regions are present in one or two copies per haploid rat genome. Northern blot analysis of RNA has shown that the rat homeo-box sequences are expressed in a tissue-specific manner; transcripts were detected in the spinal cord and kidney, but not in brain, testis, liver, and spleen. The rat nucleotide sequences lying outside the 180-nt homeo-box domain share virtually no sequence homology with the Antp flanking regions. However, one clone does show an equally high degree of amino acid homology within the homeo-box and its immediate flanking region with a putative homologous gene in mouse. The result suggests that some of the mammalian homeo-box-containing genes are conserved in evolution and may serve important cellular or developmental functions.

Tissue coenzyme Q (ubiquinone) and protein concentrations over the life span of the laboratory rat.

The coenzyme Q (ubiquinone) concentrations of a number of tissues have been determined over the life span of the male laboratory rat. Coenzyme Q increased between 2 and 18 months and decreased significantly at 25 months in the heart and kidney, and the gastrocnemius, oblique and deep aspect (red) vastus lateralis muscles. The coenzyme Q concentration of liver increased over the life span, while it remained relatively constant in brain, lung, and the superficial aspect (white) of the vastus lateralis muscle. Data are also included for organ weights and protein contents of tissues over the life span. The various roles of coenzyme Q in cellular electron transfer and its regulation, energy conservation in oxidative phosphorylation, and its clinical efficacy in diseases of energy metabolism are discussed. It is hypothesized that coenzyme Q serves as a free radical quencher in the mitochondrion, a major site of free radical formation, in addition to its other roles in cellular energy metabolism, and that its cellular diminution may contribute to the loss of cellular function accompanying ageing.

The natural history of benign lymphoepithelial lesion of the salivary gland in which there is a monoclonal population of B cells. A report of two cases.

In two patients, a diagnosis of benign lymphoepithelial lesion of the salivary gland was followed by the development of extrasalivary gland lymphoma after 10- and 9-year intervals, respectively. On review, immunohistochemistry revealed immunoglobulin light-chain restriction in the initial biopsy in each case and there was both morphological and immunohistochemical evidence linking the extrasalivary gland lymphoma with the initial lesion. It is argued that in the presence of a monoclonal B-cell population, a diagnosis of benign lymphoepithelial lesion is inappropriate. These patients fulfill the criteria for a diagnosis of low-grade B-cell lymphoma of mucosa-associated lymphoid tissue and should be treated accordingly.

Relationship between 7,12-dimethyl- and 7,8,12-trimethylbenz[a]anthracene DNA adduct formation in hematopoietic organs and leukemogenic effects.

The leukemogens 7,12-dimethylbenz[a]anthracene (DMBA) and 7,8,12-trimethylbenz[a]anthracene (TMBA) bind covalently in vivo to DNA of Long-Evans rats in the hematopoietic organs, spleen and bone marrow, and in the liver, a non-target organ. Both TMBA and DMBA depleted bone marrow cells and both agents bound persistently to the DNA of bone marrow and of liver, and less to that of spleen. The three main DMBA:deoxyribonucleoside adducts in spleen, bone marrow and liver were the same as those found previously in the liver (Dipple et al. (1983) Cancer Res., 43, 4132). There were no organ-specific or age-dependent differences in the relative amounts of adducts formed. There appears to be no direct correlation between the susceptibility of an organ to carcinogenesis and the nature and relative amount of the specific adducts formed, at least for the three organs studied here.

Decreased hepatic microsomal cytochrome P450 due to indomethacin: protective roles of 16,16-dimethylprostaglandin F2 alpha and inducing agents.

Indomethacin administration to rats caused a dose-dependent decrease in hepatic microsomal cytochrome P450, aminopyrine N-demethylase, ethoxyresorufin O-de-ethylase and benzyloxyresorufin O-debenzylase, accompanied by selective alterations in microsomal sodium dodecylsulphate polyacrylamide gel electrophoretograms. High doses (greater than or equal to 8.5 mg/kg) caused the disappearance of certain of the SDS-PAGE proteins tentatively identified as being different forms of cyt. P450, together with either increases, decreases or no change in some of the non-cyt. P450 proteins in the electrophoretogram. Concomitant administration of 16,16-dimethylprostaglandin F2 alpha gave dose-dependent protection against the deleterious effects of indomethacin on the enzymic and electrophoretic parameters of cyt. P450, but did not prevent the changes due to indomethacin in the non-cyt. P450 proteins on the electrophoretogram. In contrast, prior phenobarbitone or 3-methylcholanthrene induction prevented the effects of indomethacin on both cyt. P450 and the other microsomal proteins. Concomitant administration of SKF-525A exacerbated the effects of indomethacin on cyt. P450 and the other proteins. Indomethacin coadministration with 3-methylcholanthrene resulted in the major 3MC-induced putative cyt. P450 apoprotein having a lower mol. wt than usual. Conversely, indomethacin did not prevent the induction by SKF-525A of a different putative cyt. P450 apoprotein, despite causing decreases in cyt. P450 as determined spectrophotometrically and enzymologically. The results indicate that indomethacin rather than one of its metabolites is responsible for the decrease in cyt. P450 and that the mechanisms of protection by prostaglandin and inducing agents are, respectively, different.

Are the decreases in hepatic cytochrome P-450 and other drug-metabolising enzymes caused by indomethacin in vivo mediated by intestinal bacterial endotoxins? 16,16-Dimethylprostaglandin F2 alpha prevents decreases in hepatic drug-metabolising enzymes due to exogenous endotoxin.

Administration of either indomethacin (8.5 mg/kg) or E. coli endotoxin (3.5 mg/kg) to rats caused significant decreases in a variety of drug-metabolising enzyme activities. Either agent markedly decreased biphenyl 4-hydroxylase by 72-80% and caused lesser decreases (21-64%) in cyt. P-450, aminopyrine N-demethylase, ethoxyresorufin O-deethylase (EROD), benzyloxyphenoxazone O-debenzylase (BPOD), cyt. b5, NADPH-cyt. c reductase, NADH-cyt. b5 reductase, epoxide hydrolase (EH) and glucuronyl transferase (GT). The decreases in GT (21-22%) were significantly less than in cyt. P-450 (45-57%). Sulphotransferase was not affected by either indomethacin or endotoxin. The overall pattern of relative decreases in the different enzymes was similar for either indomethacin or endotoxin. Four activities, however, were affected to a significantly greater extent by indomethacin than by endotoxin at 2-6 mg/kg: EROD, BPOD, cyt. b5 and EH. Additionally, hepatic glutathione was decreased by indomethacin but not by endotoxin. Indomethacin or endotoxin caused similar but not identical decreases in selected protein bands in the "cyt. P-450 region" of microsomal SDS-polyacrylamide gel electrophoretograms. Concomitant administration of 16,16-dimethylprostaglandin F2 alpha afforded significant (50-100%) protection against all the above-mentioned effects of indomethacin or endotoxin. The effects of indomethacin on cyt. P-450 were lessened by concomitant administration of a mixture of neomycin, polymyxin B and bacitracin. Throughout the study there was a close correlation between the extent of decrease in hepatic cyt. P-450 and the degree of intestinal ulceration caused by indomethacin. It was concluded that bacterial endotoxins liberated into the portal blood as a result of indomethacin-induced ulceration of the small intestine probably only partially mediated the effects of indomethacin on hepatic drug-metabolising enzymes. The protection afforded by 16,16-dimethylprostaglandin F2 alpha could have been due to both the prevention of ulceration and to a direct cytoprotective effect on the liver.

Xenobiotic-metabolizing enzyme activity in human non-small-cell derived lung cancer cell lines.

Human lung cancer cell lines in culture were investigated for the expression of monooxygenase and other xenobiotic-metabolizing enzyme activities. Two bronchiolo-alveolar carcinoma derived cell lines (NCI-H322 and NCI-H358) and two small-cell carcinoma derived cell lines (NCI-H128 and NCI-H69) were used. Previous work has shown that NCI-H322 has ultrastructural features of Clara cells while NCI-H358 shows characteristics of alveolar type II cells [Schuller et al., Proc. Am. Ass. Cancer Res. 26, 27 (1985)]. NCI-H128 and NCI-H69 show very poor differentiation of cytoplasmic organelles. Cytochrome P-450 levels were spectroscopically detectable only in NCI-H322. Both NCI-H322 and NCI-H358, but not NCI-H69 and NCI-H128, exhibited aryl hydrocarbon hydroxylase (using benzo[a] pyrene as substrate) and ethoxycoumarin O-deethylase activities. These activities were highly inducible following pretreatment with the polycyclic aromatic hydrocarbons (PAH) beta-naphthoflavone or benzo[a] anthracene. The PAH produced a 2-fold increase in spectroscopically detectable cytochrome P-450 levels in NCI-H322. Following induction, cytochrome P-450 was also spectroscopically detectable in NCI-H358. No aldrin epoxidase activity was present in either untreated or pretreated cell lines. Pretreatment with phenobarbitone or dexamethasone did not induce the aryl hydrocarbon hydroxylase activity in either NCI-H322 or NCI-H358. The ethoxycoumarin O-deethylase activity in beta-naphthoflavone-pretreated NCI-H322 and NCI-H358 was inhibited in a concentration-dependent manner by ellipticine, alpha-naphthoflavone, cimetidine or metyrapone. Untreated NCI-H322 and NCI-H358 also contained cytochrome b5, NADPH cytochrome c reductase and epoxide hydrolase activities. None of these enzyme activities measured was detectable in the untreated or pretreated small-cell derived cancer cell lines (NCI-H128 and NCI-H69). These data show that the two bronchiolo-alveolar carcinoma derived cell lines (NCI-H322 and NCI-H358) exhibit cytochrome P-448-dependent monooxygenase activity and may thus prove useful to study the processes of xenobiotic activation in human lung.