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M Pirity

Publications and source records attributed to M Pirity.

8 recordsLinked to original sources
PubMed2006

Lessons learned from Myc/Max/Mad knockout mice.

The past two decades of gene targeting experiments have allowed us to make significant strides towards understanding how the Myc/Max/Mad network influences multiple aspects of cellular behavior during development. Here we summarize the findings obtained from the myc/max/mad knockout mice generated to date, namely those in which the N-myc, c-myc, L-myc, mad1, mxi1, mad3, mnt, or max genes have been targeted. A compilation of lessons we have learned from these myc/max/mad knockout mouse models, and suggestions as to where future efforts could be focused, are also presented.

Animals↗
PubMed1998

Dissecting the role of N-myc in development using a single targeting vector to generate a series of alleles.

The N-myc proto-oncogene is expressed in many organs of the mouse embryo, suggesting that it has multiple functions. A null mutation leads to mid-gestation lethality [1-4], obscuring the later roles of the gene in organogenesis. We have generated a multi-purpose gene alteration by combining the potential for homologous and site-specific recombination in a single targeting vector, and using the selectable marker for neomycin-resistance, neo, to downregulate gene activity. This allowed us to create a series of alleles that led to different levels of N-myc expression. The phenotypes revealed a spectrum of developmental problems. The hypomorphic allele produced can be repaired in situ by Cre-recombinase-mediated DNA excision. We show here for the first time the use of a single targeting vector to generate an allelic series. This, and the possibility of subsequent lineage-specific or conditional allele repair in situ, represent new genome modification strategies that can be used to investigate multiple functions of a single gene.

Alleles↗
PubMed1998

Embryonic stem cells, creating transgenic animals.

Embryonic stem (ES) cells have afforded a means of directly modifying the mouse genome in vitro and then introducing such changes directly into animals. The advent of this technology has made the mouse the mammal of choice for mutagenesis approaches used in the study of embryonic development and disease conditions. This chapter deals with the maintenance and modification of these pluripotent cell lines and describes the routes that can be taken for their efficient introduction to the in vivo environment.

Animals↗
PubMed1996

Overexpression of P-glycoprotein in heat- and/or drug-resistant hepatoma variants.

We have earlier isolated a glucocorticoid-resistant, dedifferentiated rat hepatoma variant, the clone 2, which exhibited deficient stress activation of the major stress-inducible heat-shock protein hsp68. Multidrug-resistant variants were isolated from clone 2 cells using increasing concentrations of colchicine. The induction deficiency of hsp68 was maintained in the colchicine-resistant clone 2 cells grown for several months in the presence of 1 microgram/ml colchicine (termed as highly multidrug-resistant variant) indicating that this heat-shock protein is not involved in the multidrug resistance. No alteration of the protein synthesis pattern was observed except the strong increase of the P-glycoprotein, which correlated with high level of corresponding mRNA. Stable heat-resistant variants of clone 2 were also isolated, which showed increased drug resistance to several drugs, i.e. they became moderately multidrug-resistant. This moderate multidrug resistance of the heat-resistant variants was further increased by stepwise selection with colchicine (highly multidrug-resistant heat-resistant variants). The levels of P-glycoprotein mRNA and protein were elevated both in the heat-resistant, non drug selected, moderately drug-resistant and in heat-resistant, colchicine selected, highly drug-resistant variants. Decreased retention of antitumor drugs was observed in all multidrug-resistant variants indicating that P-glycoprotein was functional. Verapamil increased doxorubicin retention and cytotoxicity significantly. Our results showing that severely stressed hepatoma cells overexpressed the multidrug resistance gene(s) raise the possibility that the P-glycoprotein may participate in protection against environmental stress such as heat.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

The function of heat-shock proteins in stress tolerance.

We earlier demonstrated that hsp68 is deficiently induced upon stress in the glucocorticoid-resistant, dedifferentiated Reuber rat hepatoma clone 2 cells, but is strongly activated in the differentiated, glucocorticoid-sensitive Faza 967 cells from which clone 2 was derived. We used the two cell types to address the questions whether hsp68 is specifically involved in the development of thermotolerance and/or thermoresistance or drug resistance. Our experiments show that clone 2 cells were not protected from the killing effect of heat by pre-treatment with sodium arsenite, whereas Faza 967 cells were. These results strongly suggest a role of hsp68 in the development of thermotolerance in hepatoma cells. Stable heat-resistant variants of clone 2 cells were also isolated, where an increased basal expression of several hsps was observed together with the (at least partial) restoration of the heat-inducibility of hsp68. These results suggest that several hsps are needed to protect the critical biological processes at high temperature. The heat-resistant hepatoma cells also became resistant to several anticancer drugs. The multidrug resistance of the hepatoma variants correlates with the overexpression of the plasma membrane P-glycoprotein. Our results showing that severely stressed hepatoma cells overexpressed the mdr gene(s) raise the possibility that the P-gp may participate in protection against environmental stress such as heat.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Decreased stress inducibility of the HSP68 protein in a rat hepatoma variant clone.

Analysis of the stress response of closely related rat hepatoma clones revealed that the major inducible heat-shock protein 68 (HSP68) was only slightly inducible upon stress in the glucocorticoid-resistant, dedifferentiated clone-2 cells, but strongly activated in the differentiated, glucocorticoid-sensitive Faza 967 cells from which clone 2 was derived. The decreased inducibility of HSP68 in clone-2 cells was not the consequence of altered kinetics of protein synthesis recovery, was not correlated with the deficient inducibility of other major heat-shock proteins and had no effect on the heat sensitivity of the cells. This deficiency was observed after treatment with mild and strong heat and various chemicals. The results of nuclear run-on experiments suggested that the impairment of HSP68 mRNA induction most likely occurs at the transcriptional level and is probably specific for the corresponding gene. In Faza 967 and clone-2 cells, stress activated comparable levels of heat-shock-factor binding to the heat-shock element, and the expression of a reporter gene under the control of murine HSP70.1 promoter was strongly stimulated in both cells. Therefore, our results raise the possibility that the deficient stress inducibility of HSP68 is due to some specific regulation of the endogeneous HSP68 gene, rather than to a deficiency of the heat-shock factor or mutation of the corresponding gene.

Animals↗
PubMedDate not supplied

P-glycoprotein is overexpressed and functional in severely heat-shocked hepatoma cells.

BACKGROUND: Hyperthermia is used in the treatment of some human malignancies. Thermotolerance may interfere with the efficacy of hyperthermic treatment, and thermotolerant cells may also display an enhanced resistance to some anticancer drugs. We have earlier isolated stable heat-resistant rat hepatoma variants and examined whether heat resistance influenced the drug sensitivity of the cells. MATERIALS AND METHODS: Heat-resistant variants were isolated by ten repeated cycles of heat exposure at 45 degrees C for 80 min. Highly multidrug-resistant variants were isolated by stepwise selection with colchicine. RESULTS: The heat-resistant variants became moderately multidrug resistant. This resistance was further increased by stepwise selection with colchicine (highly multidrug resistant variants). The levels of P-glycoprotein were elevated both in moderately and highly drug resistant variants. Decreased retention of antitumor drugs was observed in the multidrug resistant variants, verapamil increased doxorubicin retention significantly. Estradiol was almost without effect, while tamoxifen increased the drug uptake. Amplification of the MDR gene occurred in a part of the highly multidrug resistant variants. CONCLUSIONS: Acquired stable heat resistance of cancer cells can prevent the efficacy not only of hyperthermic treatment, but also the success of chemotherapy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗