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Biomedical subjects

M T Muller

Publications and source records attributed to M T Muller.

At least 19 recordsLinked to original sources
PubMed2001

Downregulation of topoisomerase I in differentiating human intestinal epithelial cells.

To better understand the increased sensitivity of proliferating intestinal epithelial cells to topoisomerase I (topo I) poisons, we examined differentiation of a human intestinal cell line (Caco-2) in the presence of camptothecin (CPT) and its analogs irinotecan (CPT-11) and topotecan (TPT). The prodrug CPT-11 exerts its antitumor activity after transformation to SN-38. We show that cleavable complex formation in vivo (on genomic DNA) induced by CPT or SN-38 is 4- to 7-fold reduced in fully differentiated cells relative to undifferentiated cells. TPT-induced cleavable complexes, however, are reduced by 30-fold. In contrast, CPT-11-driven cleavable complexes did not change during cell differentiation. In general, cytotoxicity closely paralleled cleavable complex formation, as attested to by the four- to 6-fold decrease in cytotoxicity in fully differentiated cells treated with CPT and SN-38 compared with proliferating cells. Topo I activity and polypeptide levels decreased 4-fold over the course of differentiation. This reduction occurs as Caco-2 cells approach G(1) and simultaneously differentiate. In contrast, human diploid fibroblasts do not show a reduction in topo I when entering G(1); therefore, topo I downregulation is a differentiation-specific event in the Caco-2 cell line. Cleavable complex formation and cytotoxicity induced by CPT and SN-38 correlate with topo I level and activity in cells at different stages in their differentiation. Thus, high target levels correspond closely with drug sensitivity and since proliferating cells contain larger amounts of topo I, we conclude that epithelial crypt cells probably succumb to chemotherapy involving topo I poisons.

Caco-2 Cells↗
PubMed2001

Catalytic inhibition of DNA topoisomerase IIalpha by sodium azide.

It has been demonstrated previously that sodium azide reduces the clastogenicity of several DNA topoisomerase II (topo II) poisons in cultured mammalian cells. These studies suggested that azide may be a catalytic topo II inhibitor. Azide interferes with mitochondrial production of ATP and is also known to inhibit cellular ATPases. Since topo II requires ATP for catalytic activity (enzyme turnover), it seemed likely that interference with ATP levels or ATP catabolism was the underlying mechanism of topo II inactivation; however, this has not been examined in living cells under conditions where the endogenous topo II is active on genomic DNA. The present studies were carried out to verify that azide inhibits endogenous topo II in cells. We show that azide blocks both decatenation and relaxation activity of purified topo II in a concentration dependent manner and reduces topoII/DNA covalent complex formation in cells. From these studies, it is concluded that sodium azide catalytically inactivates topo II via an ATP-sensitive process.

Antigens, Neoplasm↗
PubMed2000

p53 dependence of topoisomerase I recruitment in vivo.

DNA damage is attended by rapid recruitment of endogenous type I topoisomerase (topo I) into covalent cleavage complexes with genomic DNA in vivo. In contrast, endogenous topoisomerase II alpha and beta are not stimulated by DNA damage. We show that topo I and p53 are able to associate at arrested topo I-genomic DNA covalent complexes in vivo, suggesting that p53 directly stimulates topo I activity and damage to the genome of the afflicted cell. Moreover, cells that express wild-type p53 are most proficient at recruiting topo I after DNA damage; however, the p53 dependence is conditional because topo I recruitment after DNA damage can be restored if p53 mutant cells (containing a single mutant allele) are artificially held in G1. In contrast, p53 null mutants do not recruit topo I after DNA damage under any conditions (although camptothecin-dependent topo I/DNA complexes readily form in the nulls). These results show that topo I activation after DNA damage depends on the p53 status of the cell. It also depends upon the cell cycle in a way that is very different from that observed with DNA replication-dependent, camptothecin-mediated DNA breaks. The data suggest a model where p53 activates topo I, which inflicts additional genomic damage after the initial UV damage events. Topoisomerases therefore contribute to the p53 commitment to apoptosis, and topo I might assist in elimination of DNA-damaged cells as part of the cellular proofreading function inherent in the p53 pathway.

Antigens, Neoplasm↗
PubMed2000

Prevalence and content analysis of guidelines on handling requests for euthanasia or assisted suicide in Dutch nursing homes.

BACKGROUND: The growing number of requests for euthanasia or assisted suicide (EAS) makes it imperative for health care institutions, such as nursing homes, to have written guidelines on how to handle requests for EAS. The objective of this study was to determine the prevalence of EAS guidelines in Dutch nursing homes and to analyze the content. METHODS: Directors of patient care in 324 Dutch nursing homes were asked, by means of a mailed short list of questions, if they had an institutional guideline on EAS and, if so, to provide a copy. Guidelines were analyzed according to a structured list of items based on current jurisprudence, model documents, and opinions of experts. RESULTS: Of the 324 directors, 313 (97%) responded. In 58% of the nursing homes that responded, there existed written guidelines for EAS. Of those guidelines, 74% concerned EAS; in 26%, EAS was integrated in a guideline on terminal care. Of the guidelines, 165 (90%) were based on the policy that EAS is acceptable under specific conditions, and 18 (10%) banned EAS completely. Of the first-mentioned guidelines, 81% described one or more procedures for in-principle objections. In 65% of these guidelines, all official requirements for prudent practice were described. CONCLUSIONS: Despite the rapidly growing number of nursing-home guidelines on EAS and the existence of model documents, there is still considerable variation in the guidelines, and they can be improved in many aspects. A basic prerequisite is that the guidelines include all the official requirements for prudent practice.

Decision Making↗
PubMed2000

Decisions to treat or not to treat pneumonia in demented psychogeriatric nursing home patients: development of a guideline.

Non-treatment decisions concerning demented patients are complex: in addition to issues concerning the health of patients, ethical and legal issues are involved. This paper describes a method for the development of a guideline that clarifies the steps to be taken in the decision making process whether to forgo curative treatment of pneumonia in psychogeriatric nursing home patients. The method of development consisted of seven steps. Step 1 was a literature study from which ethical, juridical and medical factors concerning the patient's health and prognosis were identified. In step 2, a questionnaire was sent to 26 nursing home physicians to determine the relative importance of these factors in clinical practice. In a meeting of nine experienced physicians (step 3), the factors identified in step 2 were confirmed by most of these professionals. To prevent the final guideline being too directive, a concept guideline that included ethical and legal aspects was designed in the form of a "checklist of considerations" (step 4). Experts in the fields of nursing home medicine, ethics and law reviewed and commented on the concept guideline (step 5). The accordingly adapted "checklist of considerations" was tested in a pilot study (step 6), after which all experts endorsed the checklist (step 7). The resulting "checklist of considerations" structures the decision making process according to three primary domains: medical aspects, patient's autonomy, and patient's best interest (see annex at end of paper).

Advance Directive Adherence↗
PubMed1998

Induction of topoisomerase II-mediated DNA cleavage by a protoberberine alkaloid, berberrubine.

Topoisomerase II is the cytotoxic target for a number of clinically relevant antitumor drugs. Berberrubine, a protoberberine alkaloid which exhibits antitumor activity in animal models, has been identified as a specific poison of topoisomerase II in vitro. Topoisomerase II-mediated DNA cleavage assays showed that berberrubine poisons the enzyme by stabilizing topoisomerase II-DNA cleavable complexes. Subsequent proteinase K treatments revealed that berberrubine-induced DNA cleavage was generated solely by topoisomerase II. Topoisomerase II-mediated DNA religation with elevated temperature revealed a substantial reduction in DNA cleavage induced by berberrubine, to the extent comparable to that of other prototypical topoisomerase II poison, etoposide, suggesting that DNA cleavage involves stabilization of the reversible enzyme-DNA cleavable complex. However, the step at which berberrubine induces cleavable complex may differ from that of etoposide as revealed by the difference in the formation of the intermediate product, nicked DNA. This suggests that berberrubine's primary mode of linear formation may involve trapping nicked molecules, formed at transition from linear to covalently closed circular DNA. Unwinding of the duplex DNA by berberrubine is consistent with an intercalative binding mode for this compound. In addition to the ability to induce the cleavable complex mediated with topoisomerase II, berberrubine at high concentrations was shown to specifically inhibit topoisomerase II catalytic activity. Berberrubine, however, did not inhibit topoisomerase I at concentrations up to 240 microM. Cleavage sites induced by topoisomerase II in the presence of berberrubine and etoposide were mapped in DNA. Berberrubine induces DNA cleavage in a site-specific and concentration-dependent manner. Comparison of the cleavage pattern of berberrubine with that of etoposide revealed that they share many common sites of cleavage. Taken together, these results indicate that berberrubine represents a new class of antitumor agent which exhibits the topoisomerase II poison activity as well as catalytic inhibition activity and may have a potential clinical value in cancer treatment.

Berberine↗
PubMed1998

Ultraviolet-induced DNA damage stimulates topoisomerase I-DNA complex formation in vivo: possible relationship with DNA repair.

An antibody-based method was used to examine genomic DNA cleavage by endogenous topoisomerases in living cells. The method quantifies cleavable (covalent) complex formation in vivo after exposure to topoisomerase poisons, as reported previously (D. Subramanian et al., Cancer Res., 55: 2097-2103, 1995). Unexpectedly, exposing cells to UVB irradiation stimulated endogenous topoisomerase I-DNA covalent complex formation by as much as 8-fold, even in the absence of drugs that stabilize the cleavable complex. Covalent complexes are not a result of nonspecific UV protein-DNA cross-linking; rather, they result from the enzymatic activity of topoisomerase I on genomic DNA. Because the action of topoisomerase II on genomic DNA was not affected by UVB exposure, the observation appears to be specific for type I. Topoisomerase I is rapidly mobilized onto the genome (within 12 min after UVB exposure); however, topoisomerase I polypeptide levels did not show a corresponding increase, suggesting that preexisting enzyme is being recruited to sites of DNA damage. Complexes persist up to 5 h post-UV exposure (concurrent with the period of active DNA repair), and their formation is independent of S phase. These findings can be partially explained by the fact that in vitro topoisomerase I activity on UV-damaged DNA tends to favor formation of cleavage complexes; thus, a higher yield of covalent complexes are detected at or near cyclopyrimidine dimer lesions. Because repair-deficient cells are additionally compromised in their ability to recruit topoisomerase I, a direct role for the enzyme in DNA excision repair process in vivo is proposed that may be related to the activity of the xeroderma pigmentosum complementation group D helicase. Finally, these results collectively demonstrate that topoisomerase I is a repair-proficient topoisomerase in vivo.

DNA↗
PubMed1998

DNA topoisomerase II cleavage of telomeres in vitro and in vivo.

In this work, we have analyzed the reactivity of DNA topoisomerase II with telomeric DNA both in vitro and in vivo. Topoisomerase II cleavage reactions were performed on the tandem repeats of telomeric DNA. Analysis of this DNA on sequencing gels revealed that DNA topoisomerase II is catalytically active in cleaving the telomere DNA repeat. The topoisomerase II cleavage site is 5'TTAGG*G3' (cleavage site marked by the asterisk) and since telomere DNA is a tandem array of the above sequence, topoisomerase cleavage sites could exist every six base pairs. Detection of topoisomerase II cleavages was strongly dependent upon one specific topoisomerase II poison, etoposide (VP-16). A number of other topoisomerase II poisons were tested but did not stimulate cleavage activity at the telomere repeat. We have also analyzed the association of endogenous topoisomerase II with chromosomal telomeric DNA in HeLa cells. The in vivo complex of enzyme (ICE) bioassay was used to isolate topoisomerase II-DNA covalent complexes. In consistence with in vitro cleavage data, endogenous topoisomerase II-telomeric DNA complexes were detected in only etoposide-treated HeLa cells.

DNA↗
PubMed1998

Reduced activity of topoisomerase II in an Adriamycin-resistant human stomach-adenocarcinoma cell line.

A human stomach-adenocarcinoma cell line (MKN-45) was selected for resistance to Adriamycin by stepwise exposure to increasing concentrations of this agent. The resulting cell line (MKN/ADR) exhibited a high level of cross-resistance to topoisomerase II (topo II)-targeted drugs such as Adriamycin, mitoxantrone, and etoposide but showed no cross-resistance to other chemotherapeutic agents such as cisplatin, carboplatin, 5-fluorouracil, or mitomycin-C. P-glycoprotein encoded by the mdr-1 gene was not overexpressed in the MKN/ADR cell line. The doubling time of the MKN/ADR cell line (2.1 days) increased only slightly as compared with that of the MKN cell line (1.7 days). The patterns of cross-resistance to various chemotherapeutic agents led us to examine the cellular contents of topo II in both the drug-sensitive and the drug-resistant cells. Extractable topo II enzyme activity was 3-fold lower in MKN/ADR cells as compared with the parental MKN cells. Levels of topoisomerase I (topo I) catalytic activity were similar in both wild-type MKN and drug-resistant MKN/ADR cells. Southern-blot analysis of genomic DNA probed with topo IIalpha or IIbeta showed no sign of either gene rearrangement or hypermethylation. Northern-blot analysis revealed that both topo IIalpha and topo IIbeta mRNA transcripts were essentially identical in the MKN and MKN/ADR cells. In contrast, Western-blot analysis revealed an approximately 20-fold lower level of topo IIalpha in drug-resistant cells as compared with drug-sensitive cells, whereas topo IIbeta levels were similar in both lines. Moreover, the amount of in vivo topo IIalpha-DNA covalent complexes formed in the presence of etoposide was also approximately 20-fold lower in drug-resistant cells. No mutation was detected in the promoter region of the topo IIalpha gene in resistant cells as compared with sensitive cells. Thus, low levels of topo IIalpha polypeptide cannot be ascribed to changes in the mRNA levels. Collectively, the data suggest that a quantitative reduction in topo IIalpha may contribute to the resistance of MKN cells to Adriamycin and other topo II-targeted drugs.

Adenocarcinoma↗
PubMed1998

Molecular structure of human topoisomerase II alpha revealed by atomic force microscopy.

The entire human topoisomerase II alpha (hTopoII alpha) dimer was expressed in the yeast Saccaromyces cerevisiae, purified to homogeneity, and subjected to atomic force microscopy (AFM) under a tapping mode. Molecular images obtained exhibited a 'heart or donut-like' structure with a large axial hole. The main benefit of the application of AFM to study the hTopoII alpha is that clear images of the internal 'pore' have been achieved without crystallization, staining, or fixation of the sample. These images are consistent with the model in which topoisomerase II has a large internal gate for DNA strand passage.

Antigens, Neoplasm↗
PubMed1998

Euthanasia and assisted suicide: facts, figures and fancies with special regard to old age.

The objective of this paper is to describe the ethics and incidence of euthanasia and physician-assisted suicide (EAS) with special regard to old age. It is based on an assumption that if and when a practice of euthanasia and EAS is allowed, several vulnerable groups, including the elderly, may become a 'population at risk'. We describe some of these claims, and make an inventory of the arguments against a permissive policy concerning euthanasia and EAS which emphasise inherent dangers for the elderly. We then give an overview of the results of empirical studies about incidence of (request for) euthanasia and assisted suicide in the Netherlands, Australia, the UK, the US, Denmark and Norway. These results confirm that practitioners do receive requests for EAS and that EAS is performed in all these countries. However, there are large differences between these countries with regard to the numbers of requests and performances. Dutch findings concerning the age distribution of patients who received euthanasia or assisted suicide indicate that these procedures are rare in the elderly and in nursing homes. We conclude that, although euthanasia and assisted suicide are illegal, there is evidence that these practices occur in all countries studied. Most surveys on the incidence of euthanasia show lower figures than those in the Netherlands. Dutch studies do not provide any evidence for the elderly being in danger of becoming 'victims' of euthanasia or assisted suicide.

Aged↗
PubMed1997

Role of estrogen receptor gene demethylation and DNA methyltransferase.DNA adduct formation in 5-aza-2'deoxycytidine-induced cytotoxicity in human breast cancer cells.

The cytosine analog 5-aza-2'-deoxycytidine is a potent inhibitor of DNA methyltransferase. Its cytotoxicity has been attributed to several possible mechanisms including reexpression of growth suppressor genes and formation of covalent adducts between DNA methyltransferase and 5-aza-2'-deoxycytidine-substituted DNA which may lead to steric inhibition of DNA function. In this study, we use a panel of human breast cancer cell lines as a model system to examine the relative contribution of two mechanisms, gene reactivation and adduct formation. Estrogen receptor-negative cells, which have a hypermethylated estrogen receptor gene promoter, are more sensitive than estrogen receptor-positive cells and underwent apoptosis in response to 5-aza-2'-deoxycytidine. For the first time, we show that reactivation of a gene silenced by methylation, estrogen receptor, plays a major role in this toxicity in one estrogen receptor-negative cell line as treatment of the cells with anti-estrogen-blocked cell death. However, drug sensitivity of other tumor cell lines correlated best with increased levels of DNA methyltransferase activity and formation DNA.DNA methyltransferase adducts as analyzed in situ. Therefore, both reexpression of genes like estrogen receptor and formation of covalent enzyme. DNA adducts can play a role in 5-aza-2'-deoxycytidine toxicity in cancer cells.

Antimetabolites, Antineoplastic↗
PubMed1997

[Frequency of decisions to forgo (artificial) administration of food and fluids at life's end].

OBJECTIVE: To determine the frequency and characteristics of decisions to forgo (artificial) feeding and hydration with hastening of death as a possible result. DESIGN: Retrospective, descriptive study. SETTING: The Netherlands. METHODS: Data were collected from questionnaires mailed to physicians attending 6060 deaths identified from death certificates dating from August through November 1995 (response rate: 77%). RESULTS: Decisions to forgo feeding and hydration preceded 8% of all deaths studied; for deaths attended by nursing home physicians this percentage was 23%, for deaths attended by general practitioners and specialists it was 4%. In 68% of all these death cases, the patients had been 80 years of age or over, and 76% of them had been partly or completely incompetent. The non-treatment decision had been discussed with relatives in 82% of all cases, and in 89% of the death cases attended by nursing home physicians. These patients had had (possibly) life-shortening medication less frequently than other patients for whom a medical decision concerning the end of life had been made. CONCLUSION: Decisions to forgo (artificial) feeding and hydration were made relatively often by nursing home physicians, and rarely by general practitioners and specialists. These decisions were usually but not always made after discussion with relatives. There were no indications that these decisions entailed a great deal of suffering for the patients.

Aged↗
PubMed1997

Euthanatics: implementation of a protocol to standardise euthanatics among pharmacists and GPs.

The purpose of this study was to evaluate the implementation of a protocol to standardise euthanatics among pharmacists and general practitioners (GPs). Data over 1993 and 1994 were collected by means of an anonymous postal questionnaire sent to all pharmacists (n = 37) and all GPs (n = 283) working in the area in which the protocol was implemented. In total, 76% of the pharmacists and 63% of the GPs responded. All pharmacists and 65% of the GPs were aware of the existence of the protocol and all pharmacists and 42% of the GPs were also familiar with the content of the protocol. Both pharmacists and GPs had fairly positive attitudes towards the importance and possibility of the standardisation of euthanatics. Of the GPs who performed euthanasia or assisted with suicide during the research period, 59% made use of one or more standard packages. The majority of pharmacists and GPs were satisfied with the standard packages, and all GPs indicated that they intend to use the packages again in the future. This study shows that the implementation of standardised euthanatics was quite successful.

Adult↗
PubMed1997

The role of the nurse in active euthanasia and physician-assisted suicide.

The researchers wanted to obtain insight into the cooperation between physicians and nurses with regard to active euthanasia and physician-assisted suicide (EAS). In study I a stratified random sample of 203 clinical specialists, 152 general practitioners (GPs) and 50 nursing home physicians (NHPs) participated. In study II a random sample of 521 GPs was drawn from the province of North Holland and a random sample of 521 GPs was drawn from the rest of the Netherlands. For study III all NHPs were approached. Data were collected by means of an interview in study I. In studies II and III an anonymous, postal questionnaire was used. Approximately half of the GPs did not consult with nurses about a patient's request for EAS, the intention to administer EAS, and the actual administration. In 5% of cases, the NHPs and the specialists did not consult with nurses concerning these aspects. The GPs and NHPs indicated in 4% and 3% of the cases, respectively, that nurses administered the lethal drug(s) to the patients; the corresponding figure for the specialists was 21%. Almost all GPs and NHPs and about three-quarters of the specialists thought that nurses should never be allowed to administer EAS.

Cooperative Behavior↗
PubMed1997

Euthanasia and old age.

OBJECTIVE: to obtain insight into the relationship between euthanasia/physician-assisted suicide (EAS) and the age of patients to whom it was administered. DESIGN: a descriptive retrospective study. SAMPLE: cases of EAS reported between 1984 and 1993, in the province of North Holland in The Netherlands. These data are not available for the rest of The Netherlands. RESULTS: between 1984 and 1993, 1707 cases of EAS were reported to the Public Prosecutor in North Holland. The average age of the female patients to whom EAS was administered was 65 years; for men the average age was 62. For both men and women EAS was most frequently performed in the age-categories of 60-69 years and 70-79 years. Cancer and cerebrovascular accidents were positively related to age, while AIDS and multiple sclerosis were negatively related to age. In all physician reports the number of cases of EAS increased until the age-category of 70-79 years, after which EAS was less frequently performed by general practitioners, but more frequently by nursing-home physicians. In the age-groups of 70-79 years and 80 years and over the number of cases of EAS increased over the years. There were differences in the distribution of age between the patients to whom EAS was administered and all deaths. In the younger age-groups EAS was performed relatively more frequently, while the lowest percentage was found in the group aged 85 years and over. CONCLUSION: the suggestion that EAS is mainly performed among elderly people in The Netherlands is not supported by our study.

Age Factors↗
PubMed1997

Active voluntary euthanasia or physician-assisted suicide?

OBJECTIVE: To find out why Dutch general practitioners (GPs) and nursing home physicians (NHPs), and patients (according to their physician) opt for active voluntary euthanasia rather than for physician-assisted suicide, or vice-versa. DEFINITIONS: The following definitions were used in the study: Euthanasia is the intentional termination of life, by someone other than the patient, at the patient's request; physician-assisted suicide is intentionally helping a patient to terminate his or her life at his or her request. DESIGN: Two descriptive, retrospective studies. SETTING: The Netherlands. METHOD: Data were collected by means of anonymous questionnaires sent to a random sample of 521 GPs from the province of North Holland, 521 GPs from the rest of the Netherlands, and all 713 NHPs who were members of the Dutch Association of Nursing Home Physicians. Data were collected over the period 1986-1989 (inclusive) for GPs and the period 1986-June 1990 (inclusive) for NHPs. RESULTS: Forty-eight percent of the Gps, 78% of the NHPs, and about half of the patients who opted for euthanasia did so because of the physical condition of the patient. The reason GPs, NHPs, and patients gave most often for opting for physician-assisted suicide was that they wanted 'as far as possible to let the patient bear the responsibility.' CONCLUSION: In 38% of all cases for GPs and 57% of all cases for NHPs, only active voluntary euthanasia could be performed because of the patient's condition. In the other cases, where there was a choice, most GPs performed euthanasia, while most NHPs assisted in suicide. Active voluntary euthanasia was chosen primarily for medico-technical reasons, whereas physician-assisted suicide was selected primarily for moral reasons.

Adult↗