A proposed methodology for uncertainty extraction and verification in priority setting partnerships with the James Lind Alliance: an example from the Common Conditions Affecting the Hand and Wrist Priority Setting Partnership.

BACKGROUND: To report our recommended methodology for extracting and then confirming research uncertainties - areas where research has failed to answer a research question - derived from previously published literature during a broad scope Priority Setting Partnership (PSP) with the James Lind Alliance (JLA). METHODS: This process was completed in the UK as part of the PSP for "Common Conditions Affecting the Hand and Wrist", comprising of health professionals, patients and carers and reports the data (uncertainty) extraction phase of this. The PSP followed the robust methodology dictated by the JLA and sought to identify knowledge gaps, termed "uncertainties" by the JLA. Published Cochrane Systematic Reviews, Guidelines and Protocols, NICE (National Institute for Health and Care Excellence) Guidelines, and SIGN (Scottish Intercollegiate Guidelines Network) Guidelines were screened for documented "uncertainties". A robust method of screening, internally verifying and then checking uncertainties was adopted. This included independent screening and data extraction by multiple researchers and use of a PRISMA flowchart, alongside steering group consensus processes. Selection of research uncertainties was guided by the scope of the Common Conditions Affecting the Hand and Wrist PSP which focused on "common" hand conditions routinely treated by hand specialists, including hand surgeons and hand therapists limited to identifying questions concerning the results of intervention, and not the basic science or epidemiology behind disease. RESULTS: Of the 2358 records identified (after removal of duplicates) which entered the screening process, 186 records were presented to the PSP steering group for eligibility assessment; 79 were deemed within scope and included for the purpose of research uncertainty extraction (45 full Cochrane Reviews, 18 Cochrane Review protocols, 16 Guidelines). These yielded 89 research uncertainties, which were compared to the stakeholder survey, and added to the longlist where necessary; before derived uncertainties were checked against non-Cochrane published systematic reviews. CONCLUSIONS: In carrying out this work, beyond reporting on output of the Common Conditions Affecting the Hand and Wrist PSP, we detail the methodology and processes we hope can inform and facilitate the work of future PSPs and other evidence reviews, especially those with a broader scope beyond a single disease or condition.

Priorities for research in multiple conditions in later life (multi-morbidity): findings from a James Lind Alliance Priority Setting Partnership.

INTRODUCTION: multiple conditions in later life (multi-morbidity) is a major challenge for health and care systems worldwide, is of particular relevance for older people, but has not (until recently) received high priority as a topic for research. We have identified the top 10 research priorities from the perspective of older people, their carers, and health and social care professionals using the methods of a James Lind Alliance Priority Setting Partnership. METHODS: in total, 354 participants (162 older people and carers, 192 health professionals) completed a survey and 15 older people and carers were interviewed to produce 96 'unanswered questions'. These were further refined by survey and interviews to a shortlist of 21 topics, and a mix of people aged 80+ living with three or more conditions, carers and health and social care providers to prioritised the top 10. RESULTS: the key priorities were about the prevention of social isolation, the promotion of independence and physical and emotional well-being. In addition to these broad topics, the process also identified detailed priorities including the role of exercise therapy, the importance of falls (particularly fear of falling), the recognition and management of frailty and Comprehensive Geriatric Assessment. CONCLUSION: these topics provide a unique perspective on research priorities on multiple conditions in later life and complement existing UK and International recommendations about the optimisation of health and social care systems to deliver essential holistic models of care and the prevention and treatment of multiple co-existing conditions.

Setting the top 10 research priorities to improve the health of people with Type 2 diabetes: a Diabetes UK-James Lind Alliance Priority Setting Partnership.

AIMS: To describe processes and outcomes of a priority setting partnership to identify the 'top 10 research priorities' in Type 2 diabetes, involving people living with the condition, their carers, and healthcare professionals. METHODS: We followed the four-step James Lind Alliance Priority Setting Partnership process which involved: gathering uncertainties using a questionnaire survey distributed to 70 000 people living with Type 2 diabetes and their carers, and healthcare professionals; organizing the uncertainties; interim priority setting by resampling of participants with a second survey; and final priority setting in an independent group of participants, using the nominal group technique. At each step the steering group closely monitored and guided the process. RESULTS: In the first survey, 8227 uncertainties were proposed by 2587 participants, of whom 18% were from black, Asian and minority ethnic groups. Uncertainties were formatted and collated into 114 indicative questions. A total of 1506 people contributed to a second survey, generating a shortlist of 24 questions equally weighted to the contributions of people living with diabetes and their carers and those of healthcare professionals. In the final step the 'top 10 research priorities' were selected, including questions on cure and reversal, risk identification and prevention, and self-management approaches in Type 2 diabetes. CONCLUSION: Systematic and transparent methodology was used to identify research priorities in a large and genuine partnership of people with lived and professional experience of Type 2 diabetes. The top 10 questions represent consensus areas of research priority to guide future research, deliver responsive and strategic allocation of research resources, and improve the future health and well-being of people living with, and at risk of, Type 2 diabetes.

Top ten research priorities for spinal cord injury: the methodology and results of a British priority setting partnership.

STUDY DESIGN: This is a mixed-method consensus development project. OBJECTIVES: The objective of this study was to identify a top ten list of priorities for future research into spinal cord injury (SCI). SETTING: The British Spinal Cord Injury Priority Setting Partnership was established in 2013 and completed in 2014. Stakeholders included consumer organisations, healthcare professional societies and caregivers. METHODS: This partnership involved the following four key stages: (i) gathering of research questions, (ii) checking of existing research evidence, (iii) interim prioritisation and (iv) a final consensus meeting to reach agreement on the top ten research priorities. Adult individuals with spinal cord dysfunction because of trauma or non-traumatic causes, including transverse myelitis, and individuals with a cauda equina syndrome (henceforth grouped and referred to as SCI) were invited to participate in this priority setting partnership. RESULTS: We collected 784 questions from 403 survey respondents (290 individuals with SCI), which, after merging duplicate questions and checking systematic reviews for evidence, were reduced to 109 unique unanswered research questions. A total of 293 people (211 individuals with SCI) participated in the interim prioritisation process, leading to the identification of 25 priorities. At a final consensus meeting, a representative group of individuals with SCI, caregivers and health professionals agreed on their top ten research priorities. CONCLUSION: Following a comprehensive, rigorous and inclusive process, with participation from individuals with SCI, caregivers and health professionals, the SCI research agenda has been defined by people to whom it matters most and should inform the scope and future activities of funders and researchers for the years to come. SPONSORSHIP: The NIHR Oxford Biomedical Research Centre provided core funding for this project.

Complete reversal of fatal pulmonary hypertension in rats by a serine elastase inhibitor.

Progression of pulmonary hypertension is associated with increased serine elastase activity and the proteinase-dependent deposition of the extracellular matrix smooth muscle cell survival factor tenascin-C (refs. 1,2). Tenascin-C amplifies the response of smooth muscle cells to growth factors, which are also liberated through matrix proteolysis. Recent organ culture studies using hypertrophied rat pulmonary arteries have shown that elastase inhibitors suppress tenascin-C and induce smooth muscle cell apoptosis. This initiates complete regression of the hypertrophied vessel wall by a coordinated loss of cellularity and extracellular matrix. We now report that elastase inhibitors can reverse advanced pulmonary vascular disease produced in rats by injecting monocrotaline, an endothelial toxin. We began oral administration of the peptidyl trifluoromethylketone serine elastase inhibitors M249314 or ZD0892 21 days after injection of monocrotaline. A 1-week treatment resulted in 92% survival, compared with 39% survival in untreated or vehicle-treated rats. Pulmonary artery pressure and muscularization were reduced by myocyte apoptosis and loss of extracellular matrix, specifically elastin and tenascin-C. After 2 weeks, pulmonary artery pressure and structure normalized, and survival was 86%, compared with 0% in untreated or vehicle-treated rats. Although concomitant treatment with various agents can reduce pulmonary hypertension, we have documented complete regression after establishment of malignant monocrotaline-induced disease.

Partnering with survivors & families to determine research priorities for adult out-of-hospital cardiac arrest: A James Lind Alliance Priority Setting Partnership.

BACKGROUND: Research priority setting in health care has historically been done by expert health care providers and researchers and has not involved patients, family or the public. Survivors & family members have been particularly absent from this process in the field of resuscitation research and specifically adult out of hospital cardiac arrest (OHCA). As such, we sought to conduct a priority setting exercise in partnership with survivors, lay responders and their families in order to ensure that their priorities were visible. We partnered with the James Lind Alliance (UK) and used their commonly used consensus methodology for Public Priority Setting Partnerships (PSPs) to identify research priorities that reflected the perspectives of all stakeholders. METHODS: We used two rounds of public and health care professional surveys to create the initial priority lists. The initial survey collected open-ended questions while the second round consolidated the list of initial questions into a refined list for prioritization. This was done by reviewing existing evidence and thematic categorization by the multi-disciplinary steering committee. An in-person consensus workshop was conducted to come to consensus on the top ten priorities from all perspectives. The McMaster PPEET tool was used to measure engagement. RESULTS: The initial survey yielded more than 425 responses and 1450 "questions" from survivors and family members (18%), lay responders, health care providers and others. The second survey asked participants to rank a short list of 125 questions. The final top 25 questions were brought to the in-person meeting, and a top ten were selected through the JLA consensus process. The final list of top ten questions included how to improve the rate of lay responder CPR, what interventions used at the scene of an arrest can improve resuscitation and survival, how survival can be improved in rural areas of Canada, what resuscitation medications are most effective, what care patient's family members need, what post-discharge support is needed for survivors, how communication should work for everyone involved with a cardiac arrest, what factors best predict neurologically intact survival, whether biomarkers/genetic tests are effective in predicting OHCA and more research on the short and long-term psycho-social impacts of OHCA on survivors. The PPEET showed overwhelmingly positive results for the patient and family engagement experience during the final workshop. CONCLUSIONS: This inclusive research priority setting provides essential information for those doing resuscitation research internationally. The results provide a guide for priority areas of research and should drive our community to focus on questions that matter to survivors and their families in our work. In particular the Canadian Resuscitation Outcomes Consortium will be incorporating the top ten list into its strategic plan for the future.

Immunochemical studies of foot-and-mouth disease. V. Antigenic variants of virus demonstrated by immunodiffusion analyses with 19S but not 7S antibodies.

Three antigenic variants of foot-and-mouth disease virus, type A, strain 119, were demonstrated in Ouchterlony analyses utilizing serum collected from guinea pigs 7 days postinfection (DPI). Such antisera contain antibodies of the 19S class. Guinea pig antisera that contained antibodies of the 7S class were unable to distinguish between the antigenic variants. Similarly, 19S antibody was able to demonstrate antigenic differences in trypsin- and chymotrypsin-treated viruses that were not detected by 7S antibody-containing antisera. One of the antigenic variants of virus is apparently the wild type and is tentatively considered to have two antigenic determinant groupings termed the a- and b-sites (140S-ab). The 140S-ab variant was the sole or predominant antigenic type produced in guinea pigs and in large plaque-forming- and tissue culture-low passage sources of the virus. Another antigenic variant appears to possess only the b-site (140S-b) and was the major constituent in tissue culture-high passage virus preparations. The third variant, a small plaque former, was also devoid of the a-site and contains an antigenic determinant that is related to, but not identical with, the b-site. This variant appears to be a minor constituent of tissue culture-high passage virus. 7-DPI serum could be absorbed with a suitable concentration of tissue culture-high passage virus to remove antibody reactive with the b-determinant site. This absorbed serum still precipitated 140S-ab virus by virtue of still containing antibody reactive with the a-determinant site; however, the neutralizing activity was eliminated. This suggests that the b-site is critical with respect to neutralization while the a-site is noncritical.

Irradiation-induced G2/M checkpoint response requires ERK1/2 activation.

Following DNA damage, cells undergo G2/M cell cycle arrest, allowing time for DNA repair. G2/M checkpoint activation involves activation of Wee1 and Chk1 kinases and inhibition of Cdc25A and Cdc25C phosphatases, which results in inhibition of Cdc2 kinase. Results presented in this report indicate that gamma-irradiation (IR) exposure of MCF-7 cells resulted in extracellular signal regulated protein kinase 1 and 2 (ERK1/2) activation and induction of G2/M arrest. Furthermore, inhibition of ERK1/2 signaling resulted in >or=85% attenuation in IR-induced G2/M arrest and concomitant diminution of IR-induced activation of ataxia telangiectasia mutated- and rad3-related (ATR), Chk1 and Wee1 kinases as well as phosphorylation of Cdc25A-Thr506, Cdc25C-Ser216 and Cdc2-Tyr15. Moreover, incubation of cells with caffeine, which inhibits ataxia telangiectasia mutated (ATM)/ATR, or transfection of cells with short interfering RNA targeting ATR abrogated IR-induced Chk1 phosphorylation and G2/M arrest but had no effect on IR-induced ERK1/2 activation. In contrast, inhibition of ERK1/2 signaling resulted in marked attenuation in IR-induced ATR activity with little, if any, effect on IR-induced ATM activation. These results implicate IR-induced ERK1/2 activation as an important regulator of G2/M checkpoint response to IR in MCF-7 cells.

Elastase and matrix metalloproteinase inhibitors induce regression, and tenascin-C antisense prevents progression, of vascular disease.

Increased expression of the glycoprotein tenascin-C (TN) is associated with progression of clinical and experimental pulmonary hypertension. In cultured smooth muscle cells (SMCs) TN is induced by matrix metalloproteinases (MMPs) and amplifies the proliferative response to growth factors. Conversely, suppression of TN leads to SMC apoptosis. We now report that hypertrophied rat pulmonary arteries in organ culture, which progressively thicken in association with cell proliferation and matrix accumulation, can be made to regress by inhibiting either serine elastases or MMPs. This effect is associated with reduced TN, suppression of SMC proliferation, and induction of apoptosis. Selective repression of TN by transfecting pulmonary arteries with antisense/ribozyme constructs also induces SMC apoptosis and arrests progressive vascular thickening but fails to induce regression. This failure is related to concomitant expansion of a SMC population, which produces an alternative cell survival alpha(v)beta(3) ligand, osteopontin (OPN), in response to pro-proliferative cues provided by a proteolytic environment. OPN rescues MMP inhibitor-induced SMC apoptosis, and alpha(v)beta(3) blockade induces apoptosis in hypertrophied arteries. Our data suggest that proteinase inhibition is a novel strategy to induce regression of vascular disease because this overcomes the pluripotentiality of SMC-matrix survival interactions and induces coordinated apoptosis and resorption of matrix.

Stable transfectants of human MCF-7 breast cancer cells with increased levels of the human folate receptor exhibit an increased sensitivity to antifolates.

A major problem in cancer therapy is tumor drug resistance such as is found with antifolates (e.g., methotrexate [MTX]). We are specifically interested in the role of the human folate receptor (hFR) in MTX resistance. To investigate whether transfection of hFR results in increased MTX uptake and increased drug sensitivity, human mammary carcinoma (MCF-7) cells and Chinese hamster ovary cells (CHO) (cells which do not express detectable levels of hFR) were transfected with hFR cDNA. Stable human mammary carcinoma and Chinese hamster ovary transfectants expressing high levels of hFR were selected for further analysis. Transfected cells which express increased levels of hFR grow more rapidly than mock transfected or wild type cells in media containing physiologic concentrations of folates. The hFR expressed by these cells is sorted to the plasma membrane and is functional as determined by cell surface binding of a radiolabeled folic acid derivative and by internalization of [3H]methotrexate. The stable transfectants that express increased levels of hFR are also more sensitive to MTX in physiologic concentrations of folates. We conclude that increased expression of hFR by human mammary carcinoma and Chinese hamster ovary cells cultured under these conditions results in an enhanced growth rate, increased folic acid binding, and increased MTX uptake and cytotoxicity.

5' Nucleotide sequences influence serum-modulated expression of a human dihydrofolate reductase minigene.

Human dihydrofolate reductase (DHFR) gene sequences were isolated from DHFR gene-amplified breast cancer cell line MCF-7. These genomic sequences plus human DHFR cDNA sequences were used to construct a DHFR minigene. Calcium phosphate-mediated transfer of minigene DNA into DHFR gene-deleted Chinese hamster ovary cells converted these cells to a DHFR+ phenotype at a frequency of 0.12%. Minigene-transfected cells contained 20 to 30 minigene copies per cell and had DHFR enzyme levels similar to those of wild-type MCF-7 human cells (1.4 pmol/mg of protein). In contrast to gene-amplified MCF-7 cells, which contained multiple DHFR mRNA species (1.1, 1.6, 3.8, and 5.3 kilobases), only a single 3.8-kilobase DHFR mRNA was found in minigene-transfected cells. Previous studies on normal cells demonstrated modulation of DHFR levels by a variety of conditions which altered cell growth. When cell growth was induced in minigene-transfected cells by release from serum deprivation and DHFR levels were assayed at the time of maximum DNA synthesis, these levels were increased 2.4 to 3.7-fold. In contrast, the DHFR levels in cells transfected with a construct made from DHFR cDNA and viral promoter, intron, and termination sequences were unchanged. Minigene deletions were made and analyzed to determine the DHFR gene sequences responsible for regulation. Deletion of sequences upstream from 322 base pairs 5' to the start of transcription or 90 base pairs downstream from the termination of translation (which removed most of the 3' nontranslated region of the gene) did not alter the responsiveness of minigene-transfected cells to serum deprivation. However, when sequences between 322 and 113 base pairs 5' to the start of transcription were deleted, serum-dependent expression in minigene-transfected cells was affected.

Multidrug resistance.

The ability of malignant cells to develop resistance to cytotoxic drugs poses a major obstacle to the ultimate success of cancer therapy. While some mechanisms of resistance allow cells to survive exposure to a single agent, the phenomenon of multidrug resistance (MDR) confers upon cells the ability to withstand exposure to lethal doses of many structurally unrelated antineoplastic agents. MDR has been strongly linked to the overexpression of a membrane-associated glycoprotein, P-glycoprotein, which appears to play a role in drug efflux. However, several lines of evidence suggest that other mechanisms of resistance are involved in MDR; biochemical similarities observed in a human breast cancer cell line after the acquisition of MDR and in carcinogen-induced rat preneoplastic hepatic nodules indicate that changes in regulation of phase I and phase II drug-metabolizing enzymes may also play a role in MDR. An atypical pattern of MDR has been characterized and related to altered topoisomerase activity. Improvement in current cancer chemotherapy may be achieved by interfering with the regulation and expression of mechanisms of MDR.

Hormone-dependent regulation of BRCA1 in human breast cancer cells.

BRCA1 mRNA and protein levels are regulated by the steroid hormones estrogen and progesterone in human breast cancer cells. BRCA1 mRNA and protein levels were significantly decreased in estrogen-depleted MCF-7 and BT20T cells and increased again after stimulation with beta-estradiol. The increase in BRCA1 expression upon stimulation with estrogen was not coordinated with the early induction of the estrogen-dependent pS2 gene but closely paralleled the delayed increase in the S-phase dependent marker cyclin A. T47-D cells deprived of steroid hormones and subsequently stimulated with progesterone also showed a delayed increase in BRCA1 mRNA expression. However, no change in BRCA1 protein was detected in these cells. When considered together, the data suggest that steroid hormones may affect BRCA1 expression indirectly by altering the proliferative status of the cells rather than acting directly on DNA sequences in the BRCA1 gene itself.

Cross-resistance to camptothecin analogues in a mitoxantrone-resistant human breast carcinoma cell line is not due to DNA topoisomerase I alterations.

We have previously described a mitoxantrone-resistant human breast carcinoma cell line, MCF7/MX, in which resistance was associated with a defect in the energy-dependent accumulation of mitoxantrone in the absence of P-glycoprotein overexpression (M. Nakagawa et al., Cancer Res. 52: 6175-6181, 1992). We now report that this cell line is highly cross-resistant to the camptothecin analogues topotecan (180-fold), 9-aminocamptothecin (120-fold), CPT-11 (56-fold), and SN38 (101-fold), but is only mildly cross-resistant to the parent compound camptothecin (3.2-fold) and 10,11-methylenedioxy-camptothecin (2.9-fold). Topotecan accumulation was decreased in MCF7/MX cells compared to parental MCF7/WT cells, and there was a corresponding reduction in topotecan-mediated stimulation of the enzyme/DNA complex formation in MCF7/MX cells compared to MCF7/WT cells. No overexpression of the multidrug resistance-associated protein was detected compared to parental MCF7/WT cells. Furthermore, both sensitive MCF7/WT and mitoxantrone-resistant MCF7/MX cells contain equal amounts of DNA topoisomerase I protein, and DNA relaxation activities were equal in both cell lines and inhibited to the same extent by topotecan and camptothecin. Thus, these results suggest a novel mechanism of resistance to topoisomerase I inhibitors in cancer cells.

Effects of estrogen and tamoxifen on the regulation of dihydrofolate reductase gene expression in a human breast cancer cell line.

We have studied the effects of estrogen and the antiestrogen tamoxifen on the regulation of dihydrofolate reductase (DHFR) gene expression in a methotrexate-resistant (MTXR) human breast cancer cell line MCF-7, which contains a 50-fold increase in the level of DHFR enzyme and amplified DHFR gene sequences. Despite their selection for methotrexate resistance, the MTXR cells have retained many characteristics of the parental MCF-7 cell line. Concentrations of estrogen receptors as well as their binding affinity to estradiol are identical in both cell lines. MTXR MCF-7 cells remain sensitive to estrogen and respond to estradiol with an induction of progesterone receptors, as well as increases in the rate of DNA synthesis and cell growth. Incubation of MTXR MCF-7 cells with estradiol results in an additional 1.5- to 3.0-fold increase in their already elevated level of DHFR. The hormone-induced increases in DNA synthesis and DHFR levels are similar both with respect to the time course of inductions, as well as their dose response to estradiol. However, these two estrogen-induced effects are not coupled, since the induction of DHFR occurs even in the absence of concomitant DNA synthesis. Estradiol has no effect on DHFR enzyme stability; thus, the entire effect of estrogen on DHFR levels results from the increased synthesis of this housekeeping enzyme. In contrast, treatment of MTXR MCF-7 cells with the antiestrogen tamoxifen reduces the rate of DHFR enzyme synthesis, resulting in lower cellular levels of DHFR. These MTXR MCF-7 cells represent a useful model in which to study the mechanisms involved in the modulation of DHFR gene expression by estrogen and tamoxifen. Since the level of DHFR is a critical determinant of methotrexate cytotoxicity understanding, the regulation of DHFR gene expression may have clinical implications for the use of hormonal therapy in combination with chemotherapy for the treatment of breast cancer.

Antineoplastic drug sensitivity of human MCF-7 breast cancer cells stably transfected with a human alpha class glutathione S-transferase gene.

Studies have suggested that the alpha class glutathione S-transferase (GST) may protect cells from the chemotherapeutic drugs chlorambucil and melphalan. In order to further define the function of human alpha class GST, a complementary DNA which encodes it was ligated into an expression vector under the direction of the human metallothionein-IIA promoter and stably transfected into human MCF-7 breast cancer cells in conjunction with the G418-selectable plasmid pSV2neo. Clonal cell lines were identified which expressed increased levels of GST enzyme activity (2.2- to 5.6-fold). The transfected cell lines also had increased peroxidase activity using cumene hydroperoxide as the substrate (1.9- to 3.8-fold) which is consistent with the intrinsic peroxidase activity of alpha class GSTs. Southern blot analysis indicated that genomic DNA from these cells contained a fragment indistinguishable from the transfected alpha class GST complementary DNA (850 base pairs); Northern blot analysis of total cellular RNA indicated that these cells contained appropriately sized alpha class GST RNA (980 nucleotides); and Western blot analysis indicated that, while MCF-7 cells contained no detectable alpha class GST protein, the transfected cells contained markedly elevated levels of alpha class GST but no detectable mu or pi class GST. These alpha class GST transfected cells had increased resistance to ethacrynic acid (2.1- to 3.0-fold). However, the transfected cells failed to show any increased resistance measured at the drug dosage which inhibited 50% of the colony formation to the chemotherapeutic drugs chlorambucil, melphalan, Adriamycin, or cisplatin under conditions of either continuous or 1-h drug exposure. Neither was there any change in sensitivity to the cytotoxins benzo(a)pyrene, benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide (anti), or 1-chloro-2,4-dinitrobenzene. These studies indicate that expression of this human alpha class GST by itself in MCF-7 human breast cancer cells does not confer resistance to the chemotherapeutic drugs tested under the conditions used in these studies.

Adriamycin resistance in human tumor cells associated with marked alteration in the regulation of the hexose monophosphate shunt and its response to oxidant stress.

We found that Adriamycin increased the pentose phosphate shunt activity in both Adriamycin-sensitive (WT) and Adriamycin-resistant (ADRR) human breast cancer MCF-7 cells. In contrast, hydrogen peroxide and cumene hydroperoxide markedly stimulated pentose-shunt activity in ADRR but only moderately increased the activity in WT cells. Furthermore, the altered oxidation-reduction regulation is associated with changes intrinsic to the key enzymes of the pentose-shunt pathway, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase and with glutathione peroxidase. We found the Vmax values for glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were 50- and 4-fold lower, respectively, in ADRR than WT cells and the Kms of NADP+ were 10-fold lower in ADRR than WT. The activity of glutathione reductase in ADRR is 42% of that in WT. In spite of these changes, the response of the cells to both hydrogen peroxide and organic peroxide is not limited by either the capacity of the pentose shunt or glutathione reductase, but is determined by the activity of glutathione peroxidase and a glutathione transferase which possess peroxidase activity. The kinetic properties of the glucose-6-phosphate dehydrogenase in ADRR may, however, seriously limit the activity of cytochrome P-450 reductase, a major enzyme of Adriamycin conversion to a free radical.

Reduced intracellular drug accumulation in the absence of P-glycoprotein (mdr1) overexpression in mitoxantrone-resistant human MCF-7 breast cancer cells.

A mitoxantrone-resistant human MCF-7 breast cancer subline (MCF/MX) which is approximately 4000-fold resistant to mitoxantrone was isolated by serial passage of the parental wild-type MCF-7 cells (MCF/WT) in stepwise increasing concentrations of drug. MCF/MX cells were also approximately 10-fold cross-resistant to doxorubicin and etoposide but were not cross-resistant to vinblastine. Intracellular accumulation of radiolabeled mitoxantrone was markedly reduced in MCF/MX cells relative to that in the drug-sensitive MCF/WT cells. This decrease in intracellular drug accumulation into MCF/MX cells was associated with enhanced drug efflux, which was reversed when cells were incubated in the presence of sodium azide and 2, 4-dinitrophenol, suggesting an energy-dependent process. Incubation of MCF/MX cells with verapamil did not affect either the accumulation of mitoxantrone or the level of resistance in these cells. Furthermore, RNase protection and Western blot analyses failed to detect the expression of the mdr1 RNA or P-glycoprotein, a drug efflux pump known to be associated with the development of multidrug resistance in vitro. However, a polyclonal antibody directed against a synthetic peptide corresponding to the putative ATP binding domain of P-glycoprotein reacted with two (M(r) 42,000 and 85,000) membrane proteins from MCF/MX cells which were not found in MCF/WT. Functional assays and Western blot analysis for topoisomerase II revealed no differences in topoisomerase II activity or protein levels in MCF/MX cells. Thus, resistance in this cell line is apparently associated with enhanced drug efflux involving a pathway distinct from the mdr1-encoded multidrug transporter P-glycoprotein.

Multidrug resistance-associated protein gene overexpression and reduced drug sensitivity of topoisomerase II in a human breast carcinoma MCF7 cell line selected for etoposide resistance.

A human breast cancer cell line (MCF7/WT) was selected for resistance to etoposide (VP-16) by stepwise exposure to 2-fold increasing concentrations of this agent. The resulting cell line (MCF7/VP) was 28-, 21-, and 9-fold resistant to VP-16, VM-26, and doxorubicin, respectively. MCF7/VP cells also exhibited low-level cross-resistance to 4'-(9-acridinylamino)-methanesulfon-m-anisidide, mitoxantrone, and vincristine and no cross-resistance to genistein and camptothecin. Furthermore, these cells were collaterally sensitive to the alkylating agents melphalan and chlorambucil. DNA topoisomerase II levels were similar in both wild-type MCF7/WT and drug-resistant MCF7/VP cells. In contrast, topoisomerase II from MCF7/VP cells appeared to be 7-fold less sensitive to drug-induced cleavable complex formation in whole cells and 3-fold less sensitive in nuclear extracts than topoisomerase II from MCF7/WT cells. Although this suggested that the resistant cells may contain a qualitatively altered topoisomerase II, no mutations were detected in either the ATP-binding nor the putative breakage/resealing regions of either DNA topoisomerase II alpha or II beta. In addition, the steady-state intracellular VP-16 concentration was reduced by 2-fold in the resistant cells, in the absence of detectable mdr1/P-gp expression and without any change in drug efflux. In contrast, expression of the gene encoding the MRP was increased at least 10-fold in resistant MCF7/VP cells as compared to sensitive MCF7/WT cells. These results suggest that resistance to epipodophyllotoxins in MCF7/VP cells is multifactorial, involving a reduction in intracellular drug concentration, possibly as a consequence of MRP overexpression, and an altered DNA topoisomerase II drug sensitivity.

Promoting apoptosis: a novel activity associated with the cyclin-dependent kinase inhibitor p27.

p27Kip1, a cyclin-dependent kinase inhibitor, is recognized as a negative regulator of the cell cycle. In this paper, we report that overexpression of p27Kip1 triggers apoptosis in several different human cancer cell lines. Using a recombinant adenoviral vector that expresses p27Kip1 (Adp27), we found that overexpression of p27Kip1 in MDA-MB-231 breast cancer cells induces apoptosis that was seen by a number of different techniques, including flow cytometry and in situ terminal deoxynucleotidyl transferase-mediated nick end labeling, flow cytometric assay for sub-G1 population, and 4',6-diamindino-2-phenylindole staining. Cleavage of poly(ADP-ribose) polymerase and degradation of cyclin B1, events that are known to be associated with apoptosis, were also observed following overexpression of p27Kip1. This is the first report indicating a role for p27Kip1 in induction of apoptosis.