Biomedical subjects

D Chan

Publications and source records attributed to D Chan.

At least 163 records · Page 9Linked to original sources

PubMed1990

HT1080/DR4: a P-glycoprotein-negative human fibrosarcoma cell line exhibiting resistance to topoisomerase II-reactive drugs despite the presence of a drug-sensitive topoisomerase II.

HT1080/DR4 (DR4) is a doxorubicin-resistant human fibrosarcoma line that exhibits 150-fold cross-resistance to etoposide but does not overexpress P-glycoprotein (one mechanism of multiple drug resistance). We examined another possible mechanism that could explain resistance to both doxorubicin and etoposide: a quantitative or qualitative alteration in topoisomerase II, the putative nuclear target of these agents. The amount of immunoreactive topoisomerase II present in whole-cell lysates and nuclear extracts was three- to 10-fold lower in DR4 than in HT1080 cells. However, the topoisomerase II in nuclear extracts from both lines was sensitive to the effects of amsacrine (AMSA) and etoposide. Following treatment with AMSA, etoposide, and 5-iminodaunorubicin, topoisomerase II-mediated DNA cleavage in DR4 cells and nuclei was reduced compared with cleavage in HT1080 parent cells and nuclei. The difference between the HT1080 and DR4 lines in AMSA- and 5-iminodaunorubicin-induced cleavage was similar in cells and nuclei and could be due to the lower amount of DR4 topoisomerase II. By contrast, the difference between the HT1080 and DR4 lines in etoposide-induced DNA cleavage was much greater in cells than in nuclei. This finding suggested that cytosolic factors, removed from isolated nuclei, could influence the susceptibility of intact cells to the cytotoxic and DNA-cleaving actions of etoposide. The specific activities of several antioxidant enzymes, components of the cell's defense against free-radical damage that may be produced by doxorubicin or etoposide, were significantly different in HT1080 and DR4 cytosolic extracts. These differences may constitute an additional mechanism of resistance. Regardless, the magnitude of the resistance of DR4 to doxorubicin and etoposide cannot be explained solely on the basis of a topoisomerase II-related mechanism.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1990

Regulation of procollagen synthesis and processing during ascorbate-induced extracellular matrix accumulation in vitro.

Procollagen biosynthesis and matrix deposition were studied in long-term human skin fibroblast cultures exposed to ascorbic acid. Ascorbic acid specifically stimulated types I and III collagen synthesis, reaching a maximum at day 2 and maintaining a specific high rate of production until day 10 of ascorbate exposure, after which collagen production declined. The increased level of collagen synthesis after different exposure times could also be achieved by only brief treatment (10 h) of parallel scorbutic (ascorbic-acid-deficient) cultures with ascorbic acid. This brief exposure did not result in increased collagen mRNA, thus demonstrating that the ascorbate-induced increase in collagen synthesis at all stages of ascorbic acid exposure was due to post-transcriptional mechanisms, most likely a rapid increase in type 1 collagen mRNA translational efficiency. This mechanism, rather than the transcriptional activation, was the primary response and is adequate to explain the ascorbate-induced increase in collagen synthesis. These data also demonstrate that the presence of a collagenous extracellular matrix was not involved in this collagen biosynthetic regulation. During long-term exposure (18 days) to ascorbic acid, a substantial cross-linked collagenous matrix formed, following an approximately sigmoidal time course. The most rapid matrix deposition occurred during the later days of exposure when the rate of collagen synthesis was decreasing, suggesting that the presence of a pre-existing matrix is important for further collagen accumulation. Procollagen was also efficiently processed to collagen during this phase, demonstrating that efficient procollagen processing is an important regulatory event in collagen matrix deposition.

Ascorbic Acid↗

PubMed1990

Marfan syndrome: absence of type I or III collagen structural defects in 25 patients.

The structure and metabolism of type I and III collagens were studied in fibroblast cultures and dermis from 25 unrelated patients including 23 with typical Marfan syndrome and two infants with a very severe clinical form of this syndrome. Electrophoretic analysis of collagen alpha-chains, as well as one- and two-dimensional electrophoresis of collagen cyanogen bromide peptides, failed to show any evidence of primary structure defects or overmodification of lysine residues in these collagens. The proportion of hydroxylated prolyl residues in isolated alpha 1(I) chains was also normal. There was a minimal increase in the proportion of type III collagen produced by nine cultures. The findings in this study indicate that the underlying molecular defects in the patients studied are unlikely to involve the structure of the main fibrillar type I and III collagens.

Cells, Cultured↗

PubMed1990

Neuropeptide stimulation of calcium flux in human lung cancer cells: delineation of alternative pathways.

Calcium ion flux following the administration of a series of neuropeptides, N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate, and serum was monitored by flow cytometry in selected lung and breast cancer cell lines and Chinese hamster ovary cell line CHO-K1. Calcium ion flux was monitored in individual cells by flow cytometry using the indicator indo-1 AM. Five groups of neuropeptides produced calcium flux changes in lung cancer cell lines and CHO-K1 cells but not in breast cancer cells. The peak increase in free calcium was reached within 10 sec of peptide administration and declined to resting levels in 70-120 sec. When two or more members of the same group were administered simultaneously, calcium flux changes were identical to that produced by each single peptide. When two or more members of different groups were administered simultaneously, an increased calcium release occurred. When identical peptides or peptides from the same group were administered sequentially after the return of calcium concentrations to resting values, no calcium flux resulted from the second peptide. When peptides from different active groups were administered sequentially, a new calcium flux occurred after each peptide. These data are interpreted to mean that members of each active group of peptides trigger a different calcium flux pathway. Thus, many such pathways and different metabolic states exist within the cell. Elucidation of calcium flux pathways in normal and cancer cells may lead to greater understanding of the nature of the malignant defect.

Amino Acid Sequence↗

PubMed1989

Characterization of an amsacrine-resistant line of human leukemia cells. Evidence for a drug-resistant form of topoisomerase II.

HL-60/AMSA is a human leukemia cell line that is 100 times more resistant to the cytotoxic actions of the antineoplastic, topoisomerase II-reactive DNA intercalating acridine derivative amsacrine (m-AMSA) than is its parent HL-60 line. HL-60/AMSA cells are minimally resistant to etoposide, a topoisomerase II-reactive drug that does not intercalate. Previously we showed that HL-60 topoisomerase II activity in cells, nuclei, or nuclear extracts was sensitive to m-AMSA and etoposide, while HL-60/AMSA topoisomerase II was resistant to m-AMSA but sensitive to etoposide. Now we show that purified topoisomerase II from the two cell lines exhibits the same drug sensitivity or resistance as that in the nuclear extracts although the magnitude of the m-AMSA resistance of HL-60/AMSA topoisomerase II in vitro is not as great as the resistance of the intact HL-60/AMSA cells. In addition HL-60/AMSA cells are cross-resistant to topoisomerase II-reactive intercalators from the anthracycline and ellipticine families and the pattern of sensitivity or resistance to the cytotoxic actions of the various topoisomerase II-reactive drugs is paralleled by topoisomerase II-reactive drug-induced DNA cleavage and protein cross-link production in cells and the production of drug-induced, topoisomerase II-mediated DNA cleavage and protein cross-linking in isolated biochemical systems. In addition to its lowered sensitivity to intercalators, HL-60/AMSA differed from HL-60 in 1) the susceptibility of its topoisomerase II to stimulation of DNA topoisomerase II complex formation by ATP, 2) the catalytic activity of its topoisomerase II in an ionic environment chosen to reproduce the environment found within the living cell, and 3) the observed restriction enzyme pattern on a Southern blot probed with a cDNA for human topoisomerase II. These data indicate that an m-AMSA-resistant form of topoisomerase II contributes to the resistance of HL-60/AMSA to m-AMSA and to other topoisomerase II-reactive DNA intercalating agents. The drug resistance is associated with additional biochemical and molecular alterations that may be important determinants of cellular sensitivity or resistance to topoisomerase II-reactive drugs.

Adenosine Triphosphate↗

PubMed1989

A frameshift mutation results in a truncated nonfunctional carboxyl-terminal pro alpha 1(I) propeptide of type I collagen in osteogenesis imperfecta.

A codon frameshift mutation caused by a single base (U) insertion after base pair 4088 of prepro alpha 1(I) mRNA of type I procollagen was identified in a baby with lethal perinatal osteogenesis imperfecta. The mutation was identified in fibroblast RNA by a new method that allows the direct detection of mismatched bases by chemical modification and cleavage in heteroduplexes formed between mRNA and control cDNA probes. The region of mismatches was specifically amplified by the polymerase chain reaction and sequenced. The heterozygous mutation in the amplified cDNA most likely resulted from a T insertion in exon 49 of COL1A1. The frameshift resulted in a truncated pro alpha 1(I) carboxyl-terminal propeptide in which the amino acid sequence was abnormal from Val1146 to the carboxyl terminus. The propeptide lacked Asn1187, which normally carries an N-linked oligosaccharide unit, and was more basic than the normal propeptide. The distribution of cysteines was altered and the mutant propeptide was unable to form normal interchain disulfide bonds. Some of the mutant pro alpha 1(I)' chains were incorporated into type I procollagen molecules but resulted in abnormal helix formation with over-hydroxylation of lysine residues, increased degradation, and poor secretion. Only normal type I collagen was incorporated into the extracellular matrix in vivo resulting in a tissue type I collagen content approximately 20% of that of control (Bateman, J. F., Chan, D., Mascara, T., Rogers, J. G., and Cole, W. G. (1986) Biochem. J. 240, 699-708).

Base Sequence↗

PubMed1989

Changes in collagen stability and folding in lethal perinatal osteogenesis imperfecta. The effect of alpha 1 (I)-chain glycine-to-arginine substitutions.

The effect of glycine-to-arginine mutations in the alpha 1 (I)-chain on collagen triple-helix structure in lethal perinatal osteogenesis imperfecta was studied by determination of the helix denaturation temperature and by computerized molecular modelling. Arginine substitutions at glycine residues 391 and 667 resulted in similar small decreases in helix stability. Molecular modelling suggested that the glycine-to-arginine-391 mutant resulted in only a relatively small localized disruption to the helix structure. Thus the glycine-to-arginine substitutions may lead to only a small structural abnormality of the collagen helix, and it is most likely that the over-modification of lysine, poor secretion, increased degradation and other functional sequelae result from a kinetic defect in collagen helix formation resulting from the mutation.

Arginine↗

PubMed1989

Modulation of 4'-(9-acridinylamino)methanesulfon-m-anisidide-induced, topoisomerase II-mediated DNA cleavage by gossypol.

Our earlier studies have shown that gossypol [1,1',6,6',7,7'-hexahydroxy-5,5-diisopropyl - 3,3'-dimethyl - (2,2'- binaphthalene)-8,8'-dicarboxyaldehyde], a male contraceptive, inhibits DNA synthesis by decreasing the activities of DNA polymerase alpha and beta, resulting in the arrest of cells in mid-S phase [L.J. Rosenberg, R.C. Adlakha, D.M. Desai, and P.N. Rao, Biochim. Biophys. Acta, 866: 258-267, 1986]. Now we have examined the effects of gossypol on another enzyme of importance to cellular functions, topoisomerase II (topo II). We have determined the consequences of gossypol treatment on 4'-(9-acridinylamino)methane-sulfon-m anisidide (m-AMSA)-induced topoisomerase II-mediated, protein-associated DNA cleavage using the alkaline elution technique. In HeLa cells pretreated with gossypol (3.4-17.5 microM) for 8-16 h we observed a dose- and time-dependent decrease (50-75%) in DNA cleavage compared to that quantified in cells treated with m-AMSA alone. Gossypol by itself did not induce more than 25 rad-equivalents of DNA single-strand breaks even at the highest dose tested (26 microM). [14C]m-AMSA uptake was identical in treated and untreated cells. Pretreatment of cells with another inhibitor of DNA synthesis, thymidine, which blocks cells at G1/S boundary increased the m-AMSA-induced DNA cleavage by 25%, suggesting that the effect of gossypol might be due to the arrest of cells in mid-S phase. In contrast to gossypol's effects on m-AMSA-induced DNA cleavage, m-AMSA-induced cytotoxicity was actually increased in gossypol pretreated cells. Gossypol blocked topo II strand passing activity (decatenation of kinetoplast DNA) of cellular extracts from HeLa cells. The inhibition of this activity by gossypol was synergistic with the inhibition produced by m-AMSA or etoposide. These data suggest that gossypol can both inhibit topo II catalytic activity and interfere with the stabilization of topo II-DNA complex formation by m-AMSA. These data indicate that the magnitude of m-AMSA-induced DNA cleavage may not necessarily parallel the magnitude of m-AMSA-induced cytotoxicity. The cytotoxicity data may rather be explained by an action of gossypol and m-AMSA to block topo II catalytic activity at a point in the enzyme's strand passing cycle prior to cleavage complex formation that might be particularly toxic to cells in S phase. Gossypol should therefore be useful in improving our understanding of the cellular role of topo II and the consequences of interference with topo II activity by active antineoplastic agents.

Amsacrine↗

PubMed1989

Effect of retinoic acid on DNA cleavage and cytotoxicity of topoisomerase II-reactive drugs in a human head and neck squamous carcinoma cell line.

Evidence from several in vitro systems indicates that cellular responses to DNA topoisomerase II-reactive compounds (i.e., the epipodophyllotoxins and intercalating agents) may be affected by the relative rate of proliferation. Using a human head and neck squamous carcinoma cell line 183A, we have investigated the effect of beta-all-trans-retinoic acid (RA), a substance with known antiproliferative effects, on the DNA cleavage and cytotoxic activities of etoposide and 4'-(acridinylamino)methanesulfon-m-anisidide which interact with topoisomerase II. The effect of RA treatment on the activity of X-radiation and bleomycin, both of which produce free radical mediated effects, was also examined. RA treatment (10 to 20 microM for 72 h) does not significantly influence DNA cleavage induced by X-radiation or bleomycin but decreases DNA cleavage and cytotoxicity mediated by etoposide and 4'-(acridinylamino)methanesulfon-m-anisidide. Further, this effect can be demonstrated at a dose of RA that is minimally growth inhibitory. The inhibitory effect of RA appears to be localized to the nucleus given that similar effects on drug-mediated DNA cleavage can be demonstrated in nuclei isolated from RA-treated cells. However, both drug-stimulated DNA cleavage activity and topoisomerase II catalytic activity are approximately equal in crude nuclear extracts of untreated and RA-treated cells. These data suggest that the resistance to topoisomerase II-reactive drugs induced by RA treatment of 183A cells is not mediated through a direct effect on the enzyme, but, instead, is related to other changes in the nuclear milieu occurring in the initial stages of quiescence such as altered chromatin conformation.

Amsacrine↗

PubMed1989

Identification of the gene for fly non-muscle myosin heavy chain: Drosophila myosin heavy chains are encoded by a gene family.

In contrast to vertebrate species Drosophila has a single myosin heavy chain gene that apparently encodes all sarcomeric heavy chain polypeptides. Flies also contain a cytoplasmic myosin heavy chain polypeptide that by immunological and peptide mapping criteria is clearly different from the major thoracic muscle isoform. Here, we identify the gene that encodes this cytoplasmic isoform and demonstrate that it is distinct from the muscle myosin heavy chain gene. Thus, fly myosin heavy chains are the products of a gene family. Our data suggest that the contractile function required to power myosin based movement in non-muscle cells requires myosin diversity beyond that available in a single heavy chain gene. In addition, we show, that accumulation of cytoplasmic myosin transcripts is regulated in a developmental stage specific fashion, consistent with a key role for this protein in the movements of early embryogenesis.

Amino Acid Sequence↗

PubMed1989

A base substitution in the exon of a collagen gene causes alternative splicing and generates a structurally abnormal polypeptide in a patient with Ehlers-Danlos syndrome type VII.

An unusual splicing mutation has been characterized in the pro alpha 1(I) collagen gene of a sporadic case of Ehlers-Danlos Syndrome Type VII. Cloning of primer extended cDNA in conjunction with R-looping experiments established that nearly half of the pro alpha 1(I) collagen gene transcripts are abnormally spliced, for they lack exon 6 sequences. Analysis of cloned genomic fragments revealed that one of the proband's alleles displays the substitution of an A for a G in the last nucleotide of exon 6. The change converts the normal Met (ATG) codon to Ile (ATA) and, in addition, obliterates a NcoI restriction site. The latter event was exploited to demonstrate the de novo nature of the mutation since DNA from the unaffected parents was fully digested with the enzyme, after in vitro amplification by the polymerase chain reaction. Further confirmation of the missplicing was obtained by transient expression into animal cells of allelic minigene constructs. Finally, Western blot analysis of cyanogen bromide cleaved collagen and nucleotide sequencing of appropriately selected cDNA clones demonstrated the production of relatively low amounts of correctly spliced molecules harboring the Ile substitution, as well.

Amino Acid Sequence↗

PubMed1989

The effect on the bonding strength of orthodontic brackets of fluoride application after etching.

Etching of the enamel surface in preparation for bonding orthodontic brackets is an essential part of the bonding procedure. In an attempt to alleviate concerns regarding the decalcified enamel, it has been suggested that a fluoride solution be applied to the etched surface before placing the bonding material. The purpose of this study is to test the tensile bonding strength of the composite to fluoride-treated enamel. Four groups of 10 teeth each were compared. Group I received a solution of 2% NaF in 0.1 M H3PO4 after etching; groups II and III received a solution of 4% NaF in 0.1 M H3PO4 after etching; and group IV served as the control--that is, no fluoride solution was applied to the enamel after etching. The orthodontic brackets were bonded to the teeth in the four groups with the same procedure. The 40 teeth were then placed in synthetic saliva. Groups I, II, and IV were debonded after 24 hours; group III was debonded after 7 days. The Instron Universal testing machine was used to determine the tensile bonding strength of the adhesive to the teeth. The basic statistics for the tensile strength were calculated and the analysis of variance, general linear models procedure, was used to determine whether significant differences were present among the groups. The findings indicate that the application of either 2% or 4% NaF in a 0.1 M H3PO4 solution does not significantly influence the tensile bonding strength of the adhesive material to the enamel surface.

Acid Etching, Dental↗

PubMed1989

Involvement of vimentin in the reverse transformation reaction.

An organized cytoskeleton is required for the cAMP-induced reverse transformation reaction in CHO-K1 cells. In the course of the reaction a considerable fraction of the genome changes its nuclease sensitivity. The current paper presents the following evidence that cAMP-induced phosphorylation of vimentin is an early step in this reaction complex. (i) Vimentin is only slightly phosphorylated in transformed CHO-K1 cells but is heavily phosphorylated in normal fibroblasts. (ii) cAMP addition almost triples the vimentin phosphorylation of CHO-K1 cells but does not change that of normal cells. (iii) Vimentin phosphorylation is one of the earliest phenomena to occur after addition of cAMP to CHO-K1 cells, preceding the cell-stretching reaction and other manifestations of reverse transformation. (iv) Indirect immunofluorescence experiments demonstrate that vimentin appears as a condensed mass in transformed CHO-K1 cells but cAMP addition restores the filamentous structure characteristic of the normal fibroblast. (v) Other transformed cells unresponsive to reverse transformation by cAMP failed to demonstrate increased phosphorylation of vimentin on treatment with cAMP. These results support the proposed scheme that phosphorylation of cytoskeletal elements initiates a large-scale genetic regulatory action in which a substantial change in the spectrum of genome exposure and sequestration occurs. A function for intermediate filaments in reverse transformation is implied.

Animals↗

PubMed1989

Correlation of clinical and molecular biological abnormalities in osteogenesis imperfecta.

Substitution of a glycine residue in the triple helix of the alpha 1(I) chain by either arginine, valine or alanine was associated with the type II lethal perinatal osteogenesis imperfecta phenotype. This phenotype was also produced by a frameshift mutation that resulted in an abnormal amino acid sequence of the carboxy-terminal propeptide of the pro-alpha 1(I) chain. The latter baby, however, showed some clinical and radiographic differences from the other babies with type II OI. The severity of the clinical and radiographic phenotypes are likely to be determined by both the type and site of the mutation as well as by the intra-uterine environment.

Alanine↗

PubMed1988

Effect of phorbol ester treatment on drug-induced, topoisomerase II-mediated DNA cleavage in human leukemia cells.

Tumor-promoting phorbol esters such as phorbol 12-myristate 13-acetate (PMA) induce the monocytoid differentiation of HL-60 human leukemia cells. The cellular receptor for PMA is protein kinase C. However, cellular events distal to protein kinase C phosphorylation are also critical steps toward differentiation. These events may include specific programs of oncogene transcription that have been associated with phorbol ester-induced leukemic cell differentiation. Recently, it has been found that topoisomerase II could be activated by protein kinase C-mediated serine phosphorylation and that PMA treatment of HL-60 cells enhanced extractable topoisomerase II from these cells. Additionally, topoisomerase II-reactive antineoplastic drugs could block PMA-induced differentiation of HL-60. This enzyme has been implicated in gene regulation, and drug-induced, topoisomerase II-mediated DNA cleavage sites have been identified within cellular oncogenes. Thus, topoisomerase II could play a critical role in the signal transduction cascade leading from PMA-protein kinase interaction to monocytoid differentiation. We have examined this relationship between topoisomerase II and PMA-induced differentiation through measurements of drug-induced, topoisomerase II-mediated DNA cleavage (via alkaline elution) in PMA-treated HL-60 cells. Etoposide-induced DNA cleavage was reduced 10-fold in HL-60 cells treated with 10 nM PMA for 24 h. Neither dimethyl sulfoxide (which produces granulocytoid differentiation) nor non-differentiation-inducing phorbol esters could produce this effect. The decreased cleavage was not due to a PMA-induced inhibition of cell-associated etoposide and was demonstrable in nuclei isolated from PMA-treated cells. The decrease was not simply related to decreased cellular proliferation rate as reflected in the inhibition of DNA synthesis because conditions leading to marked inhibition of DNA synthesis did not necessarily inhibit etoposide-induced DNA cleavage. By contrast, lower concentrations of PMA inhibited etoposide-mediated DNA cleavage disproportionately compared with PMA effects on DNA synthesis. Interestingly, PMA reduced cleavage induced by the topoisomerase II-reactive DNA intercalator 4'-(9-acridinylamino)methanesulfon-m-anisidide by 2-fold, suggesting that specific drug-DNA interactions could partially overcome the PMA-induced effect that resulted in decreased etoposide-induced, topoisomerase II-mediated DNA cleavage. Nuclear proteins in 0.35 M NaCl extracts from untreated or PMA-treated HL-60 cells were virtually identical in topoisomerase II activity and in topoisomerase II-associated drug sensitivity.(ABSTRACT TRUNCATED AT 400 WORDS)

Amsacrine↗

PubMed1988

Substitution of arginine for glycine 664 in the collagen alpha 1(I) chain in lethal perinatal osteogenesis imperfecta. Demonstration of the peptide defect by in vitro expression of the mutant cDNA.

Structurally abnormal type I collagen was identified in tissues and cultured fibroblasts from a case of lethal perinatal osteogenesis imperfecta. Two-dimensional gel electrophoresis of the CNBr peptides demonstrated that the alpha 1(I)CB7 peptide from the alpha 1(I) chain of type I collagen existed in a normal form and a mutant form with a more basic charge distribution (Bateman, J. F., Mascara, T., Chan, D., and Cole, W. G. (1987) J. Biol. Chem. 262, 4445-4451). Sequencing of cloned alpha 1(I) cDNAs prepared using mRNA from the patient's fibroblasts demonstrated that one clone had a single base substitution of A for G which resulted in the substitution of arginine for glycine 664 within the alpha 1(I)CB7 peptide. To determine whether this mutation was responsible for the peptide map abnormality, in vitro transcription of mRNA from the mutant cDNA was performed using an SP6 vector system. The mRNA was then translated into mutant protein in a rabbit reticulocyte lysate. Peptide analysis of the protein produced from the mutant cDNA demonstrated the same altered charge distribution of the alpha 1(I)CB7 peptide as observed with tissue- and cell-derived mutant collagen peptides. This finding confirmed that the arginine for glycine 664 sequence abnormality defined in the cDNA clone was the mutation causing the observed protein peptide map defect. This mutation is consistent with the functional abnormalities of collagen observed in this case such as reduced helical stability, reduced secretion, increased degradation, and excessive posttranslational modification of lysine.

Amino Acid Sequence↗

PubMed1988

Anion-dependent modulations of DNA topoisomerase II-mediated reactions in potassium-containing solutions.

DNA binding proteins operate in an intracellular environment of low chloride concentration, yet in vitro assays of the activities of these proteins are often performed in isotonic chloride-containing solutions. Previously, the activity of bacterial DNA-binding proteins was found to be enhanced in potassium-containing solutions in which the anion glutamate (Glu) was substituted for chloride. We have extended this observation to include eukaryotic topoisomerase I and II activities. The concentration ranges over which DNA strand passing activities of these enzymes were observed was broader in KGlu than in KCl. This was also true for the topoisomerase II-mediated DNA strand passage and antineoplastic drug-dependent DNA cleavage produced by nuclear extracts from HL-60 human leukemia cells. The rate of topoisomerase II-mediated DNA strand passage was also dependent on anion moiety and concentration in potassium-containing buffers. Drug-dependent topoisomerase II-mediated DNA cleavage in intact HL-60 cell nuclei was also anion-dependent, suggesting that anion type and concentration may influence topoisomerase II-mediated events in mammalian cells as had been described for other DNA binding proteins in prokaryotic systems. This should be considered in developing biochemical assays of topoisomerase activities to reproduce intracellular conditions.

Anions↗

PubMed1988

Comprehensive analysis of collagen metabolism in vitro using [4(3H)]/[14C]proline dual-labeling and polyacrylamide gel electrophoresis.

A method to simultaneously quantify the production, secretion, and prolyl hydroxylation of individual types of collagen in cell culture samples has been developed. Collagens were biosynthetically labeled with a mixture of [14C]proline and [4-3H]proline. The labeled collagens were isolated and their component alpha-chains were resolved by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Migration of the collagen alpha-chains was determined by fluorography, and radioactivity in excised bands was quantified by scintillation counting. [14C]Proline labeling of collagen chains was used to determine the production and secretion of the different types of collagen. The ratios of the component alpha 1(I) and alpha 2(I) chains of type I collagen were also determined in this way. Prolyl hydroxylation of collagen alpha-chains was readily determined by measurement of their 3H:14C ratios. Following 4-hydroxylation, 3H was lost from the [4-3H]proline with alteration of this ratio. This dual-labeling method is suitable for the comprehensive analysis of collagen metabolism in multiple samples.

Carbon Radioisotopes↗