- Email: [email protected]
Analysis of the human purH genomic structure, a gene encoded on human chromosome 2
POSTERABSTRACTS 133 THERAPEUTIC MECHANISMS
DEOXYMJCLEOSIDE
135 CAD OVEREXI’RESSION
ANALOGS:
Plunkett. W., Huang, P., and Gandhi, V. The Univ. of Texas M. D. Anderson Ctr, Houston, TX 77030, USA The nucleoside analogs, fludarabine and gemcitabine, are biologically active only after phosphorylation to the corresponding nucleotides by cellular enzymes. Once incorporated in to DNA, an analog typically interferes with strand elongation during replication and patch synthesis involved with DNA repair. Objectives We sought to identify mechanisms and to quantitate the actions of nucleoside analogs on DNA synthesis and to develop rationales for combinations. Methods DNA primer extension assays were employed to quantitate and compare the inhibition of DNA synthesis. Cellular systems were evaluated to relate pharmacodynamics of drug action to cell death. Results and Conclusions The incorporation of nucleotide analogs into DNA by human DNA polymerase a occurred a slower rates due that were characterized by lower V,,, and greater &values. The rate of incorporation decreased as multiple analogs were incorporated in tandem. The excision of the analogs by 3’+5’ proof-reading activities associated with DNA polymerases was greatly reduced relative to removal of deoxynucleotides, suggesting that this mechanism of repair was not effective. Both drugs inhibited ribonucleotide reductase and decreased deoxynucleotide pools. Cell death was by apoptosis as determined by multiple parameters. Treatment with aphidicolin alone had no effect on cell viability, but this blocked lethality by nucleoside analogs, indicating the critical requirement for incorporation of these drugs to evoke cell death.
134
IMP-DEIIYDROGENASE
(IMPDH),
IM’OXAN-
THINE-GUANINE
PHOSPHORIBOSYLTRANSFER-
ASE
AND
(HGPRT)
(PDES) ACID
EXPRESSION
PHOSPHODIESTERASES DURING
(MI’A)-INDUCED
HUMAN
MYCOPHENOLIC
DIFFERENTIATION
NEUROBLASTOMA
CELL
IN
LINES
C.P. Quaratino, E. Messina, G. Spoto, F. Gizzi, I. Ruffini, M. Odorisio and A. Giacomello Dipartimento di Oncologia e Neuroscienze, Istituto di Scienze Biochimiche, Universita di Chieti-ITALY Objectives To examine the kinetics of IMPDH, HGPRT, PDEs expression and the changes in intracellular GTP levels during MPA-induced LA-N-5 differentiation. Design and Methods Exponentially growing cells were treated with MPA (ranging from 5 to 50 nM) or with ethanol (0.0001% v/v) from 0 to 6 days. Media were replaced every 3 days. Cellular enzyme activities were measured on the supernatant of sonicated cells. HGPRT and IMPDH activities were measured after desalting using spectrophotometric methods. PDEs activities were determined by a HPLC procedure. Nucleotide pools were quantitated by HPLC. Cell cycle phases were measured by flow cytometry. Results During MPA-induced differentiation of LA-N-5 cells there was a 50% reduction in cellular GTP concentration and in HGPRT activity and an increase in CAMP-PDE and in cGMPPDE activities with respect to controls after 6 days. IMPDH increased during the first 3 days, but decreased thereafter. Cell cycle phases were comparable in treated and in control samples. Conclusion Reduction of intracellular GTP pools did not result in a compensatory increase of HGPRT activity and may be a critical factor in neural cell differentiation. The differentiated phenotype showed increased PDEs activities. CLINICAL
BIOCHEMISTRY,
VOLUME
30, APRIL
1997
IN MAMMALIAN
CELLS
Yu Qiu and Jeffrey N. Davidson Department of Microbiology & Immunology, Albert B. Chandler Medical Center & Lucille P. Markey Cancer Center, University of Kentucky, Lexington, KY 40536-0084, U.S.A.
OF CYTOTOXICITY
Objectives In mammalian cells, aspartate transcarbamylase is part of a trienzymatic protein called CAD. A new method is described that allows for the selection of stably transfected cells expressing high levels of CAD. Design and Methods Hamster CAD cDNA was subcloned into a bicistronic expression vector along with the neomycin resistance gene. The resulting construct was transfected into a CAD delicient hamster cell line that requires uridine for growth. Medium containing G418 and lacking uridine was used for selection. An irreversible inhibitor of aspartate transcarbamylase, N-phosphonacetyl-L-aspartate, was also used to select for colonies overexpressing CAD. Results After transfection, colonies were observed that no longer required uridine for growth. Transfectants were isolated that were resistant to 1 mM inhibitor (wiId-type cells have an LD,, of 5 FM). Some of the colonies expressed levels of CAD 10 times higher than a normal hamster cell line. Conclusion An efficient, high expression system was developed which makes studying the structure and function of CAD easier.
136
ANALYSIS
OF
STRUCTURE, CHROMOSOME
THE A
HUMAN
GENE
put-H
ENCODED
GENOMIC ON
HUMAN
2
Elizabeth A. Ray1 and G. Peter Beardsley, rics, New Haven, CT 06510
Dept. of Pediat-
Objectives Recent studies have focused on the genomic organization and potential genetic regulation of the de nouo purine nucleotide biosynthetic genes in humans. We have initiated the genomic structural determination of human purH (hpurH) with the intent of identifying potential regulatory elements. Design and Methods The full length humanpurH cDNA was used to probe a human X-genomic library via hybridisation analysis. 5’-specific and 3’-specific PCR primer sets were used to screen human BAC and YAC libraries. Additionally, PCR methods were used to amplify sequences from total genomic DNA. Southern hybridisation, PCR, restriction mapping and nucleotide sequence analysis have been used to evaluate positive clones. Results Twelve positive hpurH clones were identified. These clones contain solely the 3’ end of the full length genomic clone. Three BAC and two YAC clones have been identified. Initial analysis indicates that they may be lacking a portion of the 5’ hpurH genomic sequence as well. Several introns have thus-far been identified within hpurH. Conclusions We have isolated several genomic hpurH clones. Our initial analysis has identified two introns within the 3’ end. We are currently using genomic walking techniques to retrieve the elusive 5’ end.
137
PRE-STEADY THE ZOLE
STATE
BIFIJNCTIONAL
KINETIC HUMAN
CARBOXAMIDE
FORMYLTRANSFERASEAMP LASE
(AICARFI’/IMFCHase),
RAIIYDRO-FOLATE-REQUIRING
ANALYSIS
OF
AMINO-IMIDA-
RIBONUCLEOTIDE CYCLOIIYDROA lo-FORMYLTETENZYME
ES-
SENTIAL FOR DE NOVO PIJRINE BIOSYNTHESIS Elizabeth A. Rayl, Barbara A. Moroson, G. Peter Beardsley, and Karen S. Anderson Objectives Human PurH (hPurH) is a bifunctional protein containing both AICARFT and IMPCHase activities The order of 275
DEOXYMJCLEOSIDE
135 CAD OVEREXI’RESSION
ANALOGS:
Plunkett. W., Huang, P., and Gandhi, V. The Univ. of Texas M. D. Anderson Ctr, Houston, TX 77030, USA The nucleoside analogs, fludarabine and gemcitabine, are biologically active only after phosphorylation to the corresponding nucleotides by cellular enzymes. Once incorporated in to DNA, an analog typically interferes with strand elongation during replication and patch synthesis involved with DNA repair. Objectives We sought to identify mechanisms and to quantitate the actions of nucleoside analogs on DNA synthesis and to develop rationales for combinations. Methods DNA primer extension assays were employed to quantitate and compare the inhibition of DNA synthesis. Cellular systems were evaluated to relate pharmacodynamics of drug action to cell death. Results and Conclusions The incorporation of nucleotide analogs into DNA by human DNA polymerase a occurred a slower rates due that were characterized by lower V,,, and greater &values. The rate of incorporation decreased as multiple analogs were incorporated in tandem. The excision of the analogs by 3’+5’ proof-reading activities associated with DNA polymerases was greatly reduced relative to removal of deoxynucleotides, suggesting that this mechanism of repair was not effective. Both drugs inhibited ribonucleotide reductase and decreased deoxynucleotide pools. Cell death was by apoptosis as determined by multiple parameters. Treatment with aphidicolin alone had no effect on cell viability, but this blocked lethality by nucleoside analogs, indicating the critical requirement for incorporation of these drugs to evoke cell death.
134
IMP-DEIIYDROGENASE
(IMPDH),
IM’OXAN-
THINE-GUANINE
PHOSPHORIBOSYLTRANSFER-
ASE
AND
(HGPRT)
(PDES) ACID
EXPRESSION
PHOSPHODIESTERASES DURING
(MI’A)-INDUCED
HUMAN
MYCOPHENOLIC
DIFFERENTIATION
NEUROBLASTOMA
CELL
IN
LINES
C.P. Quaratino, E. Messina, G. Spoto, F. Gizzi, I. Ruffini, M. Odorisio and A. Giacomello Dipartimento di Oncologia e Neuroscienze, Istituto di Scienze Biochimiche, Universita di Chieti-ITALY Objectives To examine the kinetics of IMPDH, HGPRT, PDEs expression and the changes in intracellular GTP levels during MPA-induced LA-N-5 differentiation. Design and Methods Exponentially growing cells were treated with MPA (ranging from 5 to 50 nM) or with ethanol (0.0001% v/v) from 0 to 6 days. Media were replaced every 3 days. Cellular enzyme activities were measured on the supernatant of sonicated cells. HGPRT and IMPDH activities were measured after desalting using spectrophotometric methods. PDEs activities were determined by a HPLC procedure. Nucleotide pools were quantitated by HPLC. Cell cycle phases were measured by flow cytometry. Results During MPA-induced differentiation of LA-N-5 cells there was a 50% reduction in cellular GTP concentration and in HGPRT activity and an increase in CAMP-PDE and in cGMPPDE activities with respect to controls after 6 days. IMPDH increased during the first 3 days, but decreased thereafter. Cell cycle phases were comparable in treated and in control samples. Conclusion Reduction of intracellular GTP pools did not result in a compensatory increase of HGPRT activity and may be a critical factor in neural cell differentiation. The differentiated phenotype showed increased PDEs activities. CLINICAL
BIOCHEMISTRY,
VOLUME
30, APRIL
1997
IN MAMMALIAN
CELLS
Yu Qiu and Jeffrey N. Davidson Department of Microbiology & Immunology, Albert B. Chandler Medical Center & Lucille P. Markey Cancer Center, University of Kentucky, Lexington, KY 40536-0084, U.S.A.
OF CYTOTOXICITY
Objectives In mammalian cells, aspartate transcarbamylase is part of a trienzymatic protein called CAD. A new method is described that allows for the selection of stably transfected cells expressing high levels of CAD. Design and Methods Hamster CAD cDNA was subcloned into a bicistronic expression vector along with the neomycin resistance gene. The resulting construct was transfected into a CAD delicient hamster cell line that requires uridine for growth. Medium containing G418 and lacking uridine was used for selection. An irreversible inhibitor of aspartate transcarbamylase, N-phosphonacetyl-L-aspartate, was also used to select for colonies overexpressing CAD. Results After transfection, colonies were observed that no longer required uridine for growth. Transfectants were isolated that were resistant to 1 mM inhibitor (wiId-type cells have an LD,, of 5 FM). Some of the colonies expressed levels of CAD 10 times higher than a normal hamster cell line. Conclusion An efficient, high expression system was developed which makes studying the structure and function of CAD easier.
136
ANALYSIS
OF
STRUCTURE, CHROMOSOME
THE A
HUMAN
GENE
put-H
ENCODED
GENOMIC ON
HUMAN
2
Elizabeth A. Ray1 and G. Peter Beardsley, rics, New Haven, CT 06510
Dept. of Pediat-
Objectives Recent studies have focused on the genomic organization and potential genetic regulation of the de nouo purine nucleotide biosynthetic genes in humans. We have initiated the genomic structural determination of human purH (hpurH) with the intent of identifying potential regulatory elements. Design and Methods The full length humanpurH cDNA was used to probe a human X-genomic library via hybridisation analysis. 5’-specific and 3’-specific PCR primer sets were used to screen human BAC and YAC libraries. Additionally, PCR methods were used to amplify sequences from total genomic DNA. Southern hybridisation, PCR, restriction mapping and nucleotide sequence analysis have been used to evaluate positive clones. Results Twelve positive hpurH clones were identified. These clones contain solely the 3’ end of the full length genomic clone. Three BAC and two YAC clones have been identified. Initial analysis indicates that they may be lacking a portion of the 5’ hpurH genomic sequence as well. Several introns have thus-far been identified within hpurH. Conclusions We have isolated several genomic hpurH clones. Our initial analysis has identified two introns within the 3’ end. We are currently using genomic walking techniques to retrieve the elusive 5’ end.
137
PRE-STEADY THE ZOLE
STATE
BIFIJNCTIONAL
KINETIC HUMAN
CARBOXAMIDE
FORMYLTRANSFERASEAMP LASE
(AICARFI’/IMFCHase),
RAIIYDRO-FOLATE-REQUIRING
ANALYSIS
OF
AMINO-IMIDA-
RIBONUCLEOTIDE CYCLOIIYDROA lo-FORMYLTETENZYME
ES-
SENTIAL FOR DE NOVO PIJRINE BIOSYNTHESIS Elizabeth A. Rayl, Barbara A. Moroson, G. Peter Beardsley, and Karen S. Anderson Objectives Human PurH (hPurH) is a bifunctional protein containing both AICARFT and IMPCHase activities The order of 275