Lesley Sutherland

The expression of UDP-glucuronosyltransferases of the UGT1 family in human liver and kidney and in response to drugs

The expression of human UDP-glucuronosyltransferase (UGT) 1 gene family in the liver and kidney was examined using specific enzyme activity, antibodies and DNA probes for each of the four family members. Phenol UGT HP1 was expressed at a similar, relatively low, abundance in each liver and kidney whereas phenol UGT HP4 was more highly expressed in the kidney. Bilirubin UGTs (HP2 and HP3) were not detectable in the kidney and HP3 was the major isoform in the liver. The UGT activities towards certain specific substrates correlated well with the respective mRNA levels in the tissues. Bilirubin UGT HP3 was induced 2-3-fold in the livers from patients treated with phenytoin and phenobarbital. Storage of a human liver in University of Wisconsin solution which contains dexamethasone and insulin caused a large accumulation of all the UGT mRNAs, but these were not quantitatively translated into expressed UGT activities. The implications of these results are discussed.

Correlation of the clinical response to chemotherapy in breast cancer with ex vivo chemosensitivity.

Chemotherapy for breast cancer is given on the basis of empirical information from clinical trials, an approach which fails to take into account the known heterogeneity of chemosensitivity between patients. Previous attempts to determine chemosensitivity ex vivo have been disappointing, but in this study results from a newly developed tumor chemosensitivity assay (TCA) have been correlated prospectively with patient response. In this study, we have used heterogeneity data for standard regimens obtained from 116 breast TCAs to set sensitivity/resistance thresholds which were then used to interpret the results from those with known clinical responses. Assay evaluability was 97% in surgical biopsies. Clinical follow-up of stage III/ IV assessable disease was obtained from 27 breast tumors which were successfully tested for chemosensitivity, including 13 needle biopsies. The ATP-TCA assay predicted response correctly in 22 out of 29 (76%) tumors with clinically evaluable disease, suggesting that it is capable of predicting outcome in individual patients. Assays were performed in seven patients before and after chemotherapy using residual or recurrent tumor tissue. Four cases with initial sensitivity showed a decrease in sensitivity within 6 months of starting chemotherapy, while two others without clinical resistance were still sensitive by TCA. All nine courses of therapy given on the basis of TCA sensitivity resulted in partial or complete responses. Controlled trials of TCA-directed treatment against standardized empirical therapy should be conducted before this technology is widely adopted to assess its impact on rates of response, survival and the cost of treatment.

Methotrexate chemosensitivity by ATP luminescence in human leukemia cell lines and in breast cancer primary cultures: comparison of the TCA-100 assay with a clonogenic assay.

Chemosensitivity assays are widely used to predict the ability of tumor cell lines to respond to potential or existing cytotoxic drugs. In this study we have compared the cell cloning assay first described by Salmon and Hamburger with a recently developed assay which measures viable cell number by ATP luminescence. Methotrexate (MTX) was chosen as the test agent, since cell lines with varying degrees of sensitivity to this agent were readily available. The results shown good correlation between the two assays, both of which are able to discriminate between the various cell lines used. MTX inhibition of primary breast carcinomas and cell lines shows a steep dose-response curve with a threshold concentration above which increasing dose does not increase sensitivity. In solid tumors, the plateau is usually reached at a level well below 100% inhibition. The ATP luminescence assay allows discrimination of MTX sensitivity between breast carcinomas and has considerable technical advantages over the cloning assay.

The Osmotically Regulated proU Locus of Salmonella typhimurium Encodes a Periplasmic Betaine-binding Protein

The proU locus of Salmonella typhimurium encodes an osmotically induced betaine transport system. We have identified a 31 kDa periplasmic protein, encoded by proU, whose synthesis is induced by osmotic stress. A specific betaine-binding activity with a KD of about 1 microM is also present in the periplasm of osmotically induced cells. This activity is absent in those proU mutants which lack the 31 kDa periplasmic protein. Thus, ProU is a periplasmic binding-protein-dependent transport system.

Characterisation of a human bilirubin UDP-glucuronosyltransferase stably expressed in hamster lung fibroblast cell cultures

A cDNA encoding a human bilirubin UDP‐glucuronosyltransferase has been isolated and stably expressed in Chinese hamster V79 lung fibroblast cell line. Western blotting of cell homogenates with anti‐UGT antibody revealed a highly expressed protein of approx. 55.5 kDa in size. The expressed enzyme specifically catalysed the formation of bilirubin mono‐ and diglucuronides, and also catalysed the glucuronidation of two phenolic compounds, which are good substrates for other human UGT isoenzymes, at low rates.

The influence of storage on cytotoxic drug activity in an ATP-based chemosensitivity assay.

The use of viability assays to assess the effect of antineoplastic agents on cell lines and tumor cells is an important investigative tool and may have clinical relevance. Such assays require very small quantities of drugs and it is the practice of many laboratories to freeze aliquots of drugs for use in these assays as required. We have investigated the stability of 11 different agents in an ATP-based chemosensitivity assay which is being evaluated for clinical use. The results show that most drugs maintain their biological activity well when frozen at -20 degrees C for periods up to 24 months, or occasionally at room temperature. However, 4-hydroperoxycyclophosphamide and mitomycin C are exceptions to this rule, and should not be kept frozen for more than 2-3 months. Cisplatin is unstable when frozen and then thawed, but maintained activity at room temperature for at least 6 months. Since biological activity may not correlate completely with chemical stability, further studies on the effect of storage are required, but it seems unlikely that the appropriate use of frozen aliquots is a major source of error in tumor chemosensitivity assays.

The UDP-glucuronosyltransferase gene family: Function and expression of human UGTs

The UDP-glucuronosyltransferase UGTs are a group of isoenzymes of 50-60 kDa localised primarily in hepatic endoplasmic reticulum and nuclear envelope (Tephly and Burchell, 1990). The UGTs are encoded by a large multigene family, which has evolved to produce protein catalysts with different, but sometimes apparently overlapping substrate specificities. The expression of UGTs is differentially regulated by chemical and hormonal inducers and during development, thereby producing different tissue specific profiles of activities (Burchell and Coughtrie, 1989).