Biomedical subjects

F Yuan

Publications and source records attributed to F Yuan.

At least 19 recordsLinked to original sources

PubMed2000

Error-prone lesion bypass by human DNA polymerase eta.

DNA lesion bypass is an important cellular response to genomic damage during replication. Human DNA polymerase eta (Pol(eta)), encoded by the Xeroderma pigmentosum variant (XPV) gene, is known for its activity of error-free translesion synthesis opposite a TT cis-syn cyclobutane dimer. Using purified human Pol(eta), we have examined bypass activities of this polymerase opposite several other DNA lesions. Human Pol(eta) efficiently bypassed a template 8-oxoguanine, incorporating an A or a C opposite the lesion with similar efficiencies. Human Pol(eta) effectively bypassed a template abasic site, incorporating an A and less frequently a G opposite the lesion. Significant -1 deletion was also observed when the template base 5' to the abasic site is a T. Human Pol(eta) partially bypassed a template (+)-trans-anti-benzo[a]pyrene-N:(2)-dG and predominantly incorporated an A, less frequently a T, and least frequently a G or a C opposite the lesion. This specificity of nucleotide incorporation correlates well with the known mutation spectrum of (+)-trans-anti-benzo[a]pyrene-N:(2)-dG lesion in mammalian cells. These results show that human Pol(eta) is capable of error-prone translesion DNA syntheses in vitro and suggest that Pol(eta) may bypass certain lesions with a mutagenic consequence in humans.

Animals↗

PubMed2000

Error-free and error-prone lesion bypass by human DNA polymerase kappa in vitro.

Error-free lesion bypass and error-prone lesion bypass are important cellular responses to DNA damage during replication, both of which require a DNA polymerase (Pol). To identify lesion bypass DNA polymerases, we have purified human Polkappa encoded by the DINB1 gene and examined its response to damaged DNA templates. Here, we show that human Polkappa is a novel lesion bypass polymerase in vitro. Purified human Polkappa efficiently bypassed a template 8-oxoguanine, incorporating mainly A and less frequently C opposite the lesion. Human Polkappa most frequently incorporated A opposite a template abasic site. Efficient further extension required T as the next template base, and was mediated mainly by a one-nucleotide deletion mechanism. Human Polkappa was able to bypass an acetylaminofluorene-modified G in DNA, incorporating either C or T, and less efficiently A opposite the lesion. Furthermore, human Polkappa effectively bypassed a template (-)-trans-anti-benzo[a]pyrene-N:(2)-dG lesion in an error-free manner by incorporating a C opposite the bulky adduct. In contrast, human Polkappa was unable to bypass a template TT dimer or a TT (6-4) photoproduct, two of the major UV lesions. These results suggest that Polkappa plays an important role in both error-free and error-prone lesion bypass in humans.

2-Acetylaminofluorene↗

PubMed2000

Human DNA polymerase kappa synthesizes DNA with extraordinarily low fidelity.

Escherichia coli DNA polymerase IV encoded by the dinB gene is involved in untargeted mutagenesis. Its human homologue is DNA polymerase kappa (Polkappa) encoded by the DINB1 gene. Our recent studies have indicated that human Polkappa is capable of both error-free and error-prone translesion DNA synthesis in vitro. However, it is not known whether human Polkappa also plays a role in untargeted mutagenesis. To examine this possibility, we have measured the fidelity of human Polkappa during DNA synthesis from undamaged templates. Using kinetic measurements of nucleotide incorporations and a fidelity assay with gapped M13mp2 DNA, we show that human Polkappa synthesizes DNA with extraordinarily low fidelity. At the lacZalpha target gene, human Polkappa made on average one error for every 200 nucleotides synthesized, with a predominant T-->G transversion mutation at a rate of 1/147. The overall error rate of human Polkappa is 1.7-fold lower than human Poleta, but 33-fold higher than human Polbeta, a DNA polymerase with very low fidelity. Thus, human Polkappa is one of the most inaccurate DNA polymerases known. These results support a role for human Polkappa in untargeted mutagenesis surrounding a DNA lesion and in DNA regions without damage.

Base Pair Mismatch↗

PubMed2000

Specificity of DNA lesion bypass by the yeast DNA polymerase eta.

DNA polymerase eta (Pol(eta), xeroderma pigmentosum variant, or Rad30) plays an important role in an error-free response to unrepaired UV damage during replication. It faithfully synthesizes DNA opposite a thymine-thymine cis-syn-cyclobutane dimer. We have purified the yeast Pol(eta) and studied its lesion bypass activity in vitro with various types of DNA damage. The yeast Pol(eta) lacked a nuclease or a proofreading activity. It efficiently bypassed 8-oxoguanine, incorporating C, A, and G opposite the lesion with a relative efficiency of approximately 100:56:14, respectively. The yeast Pol(eta) efficiently incorporated a C opposite an acetylaminofluorene-modified G, and efficiently inserted a G or less frequently an A opposite an apurinic/apyrimidinic (AP) site but was unable to extend the DNA synthesis further in both cases. However, some continued DNA synthesis was observed in the presence of the yeast Pol(zeta) following the Pol(eta) action opposite an AP site, achieving true lesion bypass. In contrast, the yeast Pol(alpha) was able to bypass efficiently a template AP site, predominantly incorporating an A residue opposite the lesion. These results suggest that other than UV damage, Pol(eta) may also play a role in bypassing additional DNA lesions, some of which can be error-prone.

2-Acetylaminofluorene↗

PubMed2000

Vascular permeability in a human tumour xenograft: molecular charge dependence.

Molecular charge is one of the main determinants of transvascular transport. There are, however, no data available on the effect of molecular charge on microvascular permeability of macromolecules in solid tumours. To this end, we measured tumour microvascular permeability to different proteins having similar size but different charge. Measurements were performed in the human colon adenocarcinoma LS174T transplanted in transparent dorsal skinfold chambers in severe combined immunodeficient (SCID) mice. Bovine serum albumin (BSA) and IgG were fluorescently labelled and were either cationized by conjugation with hexamethylenediamine or anionized by succinylation. The molecules were injected i.v. and the fluorescence in tumour tissue was quantified by intravital fluorescence microscopy. The fluorescence intensity and pharmacokinetic data were used to calculate the microvascular permeability. We found that tumour vascular permeability of cationized BSA (pI-range: 8.6-9.1) and IgG (pI: 8.6-9.3) was more than two-fold higher (4.25 and 4.65x10(-7) cm s(-1)) than that of the anionized BSA (pI approximately 2.0) and IgG (pI: 3.0-3.9; 1.11 and 1.93x10(-7) cm s(-1), respectively). Our results indicate that positively charged molecules extravasate faster in solid tumours compared to the similar-sized compounds with neutral or negative charges. However, the plasma clearance of cationic molecules was approximately 2x faster than that of anionic ones, indicating that the modification of proteins enhances drug delivery to normal organs as well. Therefore, caution should be exercised when such a strategy is used to improve drug and gene delivery to solid tumours.

Animals↗

PubMed2000

Indole-3-carbinol is a negative regulator of estrogen receptor-alpha signaling in human tumor cells.

Estrogen, via its binding to the estrogen receptor (ER), plays an important role in breast cancer cell proliferation and tumor development. Indole-3-carbinol (I3C), a compound occurring naturally in cruciferous vegetables, exhibits a potent antitumor activity via its regulation of estrogen activity and metabolism. This study was designed to determine the effect of I3C on the potential to inhibit the ER-alpha. Using a reporter gene driven by the estrogen receptor, I3C (10-125 micromol/L) significantly repressed the 17ss-estradiol (E2)-activated ER-alpha signaling in a dose-dependent manner. I3C and breast cancer susceptibility gene 1 (BRCA1) synergistically inhibited transcriptional activity of ER-alpha. Moreover, I3C down-regulated the expression of the estrogen-responsive genes, pS2 and cathepsin-D, and up-regulated BRCA1. The inhibitory effects of I3C did not contribute to its cytotoxic effects because these activities were observed at less than toxic concentrations. These results further suggest that antitumor activities of I3C are associated not only with its regulation of estrogen activity and metabolism, but also its modulation of ER transcription activity.

Breast Neoplasms↗

PubMed2000

Mutational analysis of the Arabidopsis nucleotide binding site-leucine-rich repeat resistance gene RPS2.

Disease resistance proteins containing a nucleotide binding site (NBS) and a leucine-rich repeat (LRR) region compose the largest class of disease resistance proteins. These so-called NBS-LRR proteins confer resistance against a wide variety of phytopathogens. To help elucidate the mechanism by which NBS-LRR proteins recognize and transmit pathogen-derived signals, we analyzed mutant versions of the Arabidopsis NBS-LRR protein RPS2. The RPS2 gene confers resistance against Pseudomonas syringae strains carrying the avirulence gene avrRpt2. The activity of RPS2 derivatives in response to AvrRpt2 was measured by using a functional transient expression assay or by expressing the mutant proteins in transgenic plants. Directed mutagenesis revealed that the NBS and an N-terminal leucine zipper (LZ) motif were critical for RPS2 function. Mutations near the N terminus, including an LZ mutation, resulted in proteins that exhibited a dominant negative effect on wild-type RPS2. Scanning the RPS2 molecule with a small in-frame internal deletion demonstrated that RPS2 does not have a large dispensable region. Overexpression of RPS2 in the transient assay in the absence of avrRpt2 also led to an apparent resistant response, presumably a consequence of a low basal activity of RPS2. The NBS and LZ were essential for this overdose effect, whereas the entire LRR was dispensable. RPS2 interaction with a 75-kD protein (p75) required an N-terminal portion of RPS2 that is smaller than the region required for the overdose effect. These findings illuminate the pathogen recognition mechanisms common among NBS-LRR proteins.

Amino Acid Sequence↗

PubMed2000

Preferential incorporation of G opposite template T by the low-fidelity human DNA polymerase iota.

DNA polymerase activity is essential for replication, recombination, repair, and mutagenesis. All DNA polymerases studied so far from any biological source synthesize DNA by the Watson-Crick base-pairing rule, incorporating A, G, C, and T opposite the templates T, C, G, and A, respectively. Non-Watson-Crick base pairs would lead to mutations. In this report, we describe the ninth human DNA polymerase, Pol(iota), encoded by the RAD30B gene. We show that human Pol(iota) violates the Watson-Crick base-pairing rule opposite template T. During base selection, human Pol(iota) preferred T-G base pairing, leading to G incorporation opposite template T. The resulting T-G base pair was less efficiently extended by human Pol(iota) compared to the Watson-Crick base pairs. Consequently, DNA synthesis frequently aborted opposite template T, a property we designated the T stop. This T stop restricted human Pol(iota) to a very short stretch of DNA synthesis. Furthermore, kinetic analyses show that human Pol(iota) copies template C with extraordinarily low fidelity, misincorporating T, A, and C with unprecedented frequencies of 1/9, 1/10, and 1/11, respectively. Human Pol(iota) incorporated one nucleotide opposite a template abasic site more efficiently than opposite a template T, suggesting a role for human Pol(iota) in DNA lesion bypass. The unique features of preferential G incorporation opposite template T and T stop suggest that DNA Pol(iota) may additionally play a specialized function in human biology.

Base Pairing↗

PubMed2000

Interstitial hydraulic conductivity in a fibrosarcoma.

Convective transport of therapeutic agents in solid tumors can be improved through intratumoral infusion. To optimize the convection, we investigated the dependence of the hydraulic conductivity on tissue deformation induced by interstitial fluid pressure gradient during the infusion. Two experimental systems were used in the investigation: 1) one-dimensional perfusion through tumor slices and 2) intratumoral infusion using a needle. With these systems, we found that the apparent hydraulic conductivity (K(app)) could be altered by several orders of magnitude in fibrosarcomas through changes in perfusion conditions. When the perfusion pressure was less than a threshold level, fluid flow in tissues could not be detected. When the perfusion pressure was increased above the threshold level, K(app) depended on perfusion system and pressure. The maximum variation in K(app) in fibrosarcomas reached 80,260-fold in our experiments. The large variation in K(app) could be explained by perfusion pressure-induced tissue deformation. These experimental data suggest that the hydraulic conductivity is very sensitive to tissue deformation and imply that it is possible to improve intratumoral infusion of therapeutic agents through optimization of infusion conditions.

Animals↗

PubMed1999

The human REV1 gene codes for a DNA template-dependent dCMP transferase.

DNA is frequently damaged by various physical and chemical agents. DNA damage can lead to mutations during replication. In the yeast Saccharomyces cerevisiae, the damage-induced mutagenesis pathway requires the Rev1 protein. We have isolated a human cDNA homologous to the yeast REV1 gene. The human REV1 cDNA consists of 4255 bp and codes for a protein of 1251 amino acid residues with a calculated molecular weight of 138 248 Da. The human REV1 gene is localized between 2q11.1 and 2q11.2. We show that the human REV1 protein is a dCMP transferase that specifically inserts a dCMP residue opposite a DNA template G. In addition, the human REV1 transferase is able to efficiently and specifically insert a dCMP opposite a DNA template apurinic/apyrimidinic (AP) site or a uracil residue. These results suggest that the REV1 transferase may play a critical role during mutagenic translesion DNA synthesis bypassing a template AP site in human cells. Consistent with its role as a fundamental mutagenic protein, the REV1 gene is ubiquitously expressed in various human tissues.

Amino Acid Sequence↗

PubMed1999

Augmentation of transvascular transport of macromolecules and nanoparticles in tumors using vascular endothelial growth factor.

The goal of this investigation was to measure changes in vascular permeability, pore cutoff size, and number of transvascular transport pathways as a function of time and in response to vascular endothelial growth factor (VEGF), placenta growth factor (PIGF-1 and PIGF-2), or basic fibroblast growth factor (bFGF). Two human and two murine tumors were implanted in the dorsal skin chamber or cranial window. Vascular permeability to BSA (approximately 7 nm in diameter) and extravasation of polyethylene glycol-stabilized long-circulating liposomes (100-400 nm) and latex microspheres (approximately 800 nm) were determined by intravital microscopy. Vascular permeability was found to be temporally heterogeneous. VEGF superfusion (100 ng/ml) significantly increased vascular permeability to albumin in normal s.c. vessels, whereas a 30-fold higher dose of VEGF (3000 ng/ml) was required to increase permeability in pial vessels, suggesting that different tissues exhibit different dose thresholds for VEGF activity. Furthermore, VEGF superfusion (1000 ng/ml) increased vascular permeability to albumin in a hypopermeable human glioma xenograft in cranial window, whereas VEGF superfusion (10-1000 ng/ml) failed to increase permeability in a variety of hyperpermeable tumors grown in dorsal skin chamber. Interestingly, low-dose VEGF treatment (10 ng/ml) doubled the maximum pore size (from 400 to 800 nm) and significantly increased the frequency of large (400 nm) pores in human colon carcinoma xenografts. PIGF-1, PIGF-2, or bFGF did not show any significant effect on permeability or pore size in tumors. These findings suggest that exogenous VEGF may be useful for augmenting the transvascular delivery of larger antineoplastic agents such as gene targeting vectors and encapsulated drug carriers (typical range, 100-300 nm) into tumors.

Animals↗

PubMed1999

Available volume fraction of macromolecules in the extravascular space of a fibrosarcoma: implications for drug delivery.

Steric exclusion of molecules in the extravascular space of tissues can be quantified by the available volume fraction (K(AV)). Despite its clinical importance, however, there is a paucity of data in the literature regarding the available volume fraction of macromolecules in the extravascular space of tumor tissues. In this study, we quantified K(AV) of inulin, BSA, and dextran molecules of Mr 10,000-2,000,000 in polymer gels and fibrosarcoma tissues. The measurement involved: (a) sectioning of gels or tumor tissues into thin slices (approximately 600 microm) using a Vibratome, (b) ex vivo incubation of the slices in solutions containing fluorescently labeled tracers, and (c) quantification of the equilibrium tracer concentrations in both slices and solutions. We found that K(AV) in gels decreased monotonically when the Mr of dextran was increased from Mr 10,000 to 2,000,000. However, K(AV) in tumor tissues was insensitive to the molecular weight of dextran in the range between Mr 10,000 and 40,000. There was a sharp decrease in K(AV) from 0.28 +/- 0.14 to 0.10 +/- 0.06 when the molecular weight was increased from Mr 40,000 to 70,000. In addition to the molecular weight dependence, K(AV) was heterogeneous in tumors, with intertumoral difference being greater than intratumoral variation. The interstitial fluid space, which was quantified by K(AV) of inulin, was 50% of the total tissue volume. These data indicate that the fraction of the extravascular volume in tumors that is accessible to large therapeutic agents is heterogeneous and depends on the size of agents.

Animals↗

PubMed1999

BRCA1 inhibition of estrogen receptor signaling in transfected cells.

Mutations of the breast cancer susceptibility gene BRCA1 confer increased risk for breast, ovarian, and prostatic cancers, but it is not clear why the mutations are associated with these particular tumor types. In transient transfection assays, BRCA1 was found to inhibit signaling by the ligand-activated estrogen receptor (ER-alpha) through the estrogen-responsive enhancer element and to block the transcriptional activation function AF-2 of ER-alpha. These results raise the possibility that wild-type BRCA1 suppresses estrogen-dependent transcriptional pathways related to mammary epithelial cell proliferation and that loss of this ability contributes to tumorigenesis.

BRCA1 Protein↗

PubMed1999

Characteristics of I(K) and its response to quinidine in experimental healed myocardial infarction.

INTRODUCTION: Mechanisms and drug treatment of serious ventricular arrhythmias in patients with healed myocardial infarction (HMI) are incompletely understood, in part because the electrophysiology and pharmacology of myocytes from noninfarcted regions of HMI hearts are not well characterized. METHODS AND RESULTS: We studied the delayed rectifier potassium current (I(K)) and quinidine responsiveness of single left ventricular subendocardial myocytes isolated from the region remote to the border zone of healed infarct myocardium (4 to 6 mm from scar edge) in cat hearts 2 months after coronary artery occlusion. Subendocardial cells isolated from corresponding regions of normal cat hearts provided controls. I(K) activation and tail currents were recorded using whole cell, voltage clamp techniques. Membrane capacitance of cells remote to HMI (187 +/- 7 pF) was significantly greater than normal (155 +/- 6 pF; P < 0.001). Action potential durations (APDs) recorded from myocytes in remote regions were prolonged (APD90 = 247 +/- 10 msec) compared to normal (214 +/- 11 msec; P < 0.05). Quinidine (1 microM) significantly prolonged APD90 in normal cells but not in remote cells. Density of I(K) (tail current) was significantly decreased in remote cells (3.1 +/- 0.3 pA/pF) compared to normal (3.9 +/- 0.3 pA/pF; P < 0.05), and voltage-dependent activation of I(K) was shifted in the positive direction. Quinidine had significantly less incremental blocking effect on I(K) already blunted by regional hypertrophy compared to its effect on normal cells in remote cells. IC50 shifted to 0.95 microM in remote cells compared with 0.50 microM in normal cells. CONCLUSION: Cells in noninfarct region remote from the scar are hypertrophied and display altered electrophysiology. Their reduced I(K) responsiveness to quinidine may explain, in part, failure of quinidine to prolong APD in such cells. Moreover, dispersion of repolarization may be decreased by the effect of quinidine on normal cells.

Action Potentials↗

PubMed1999

Altered growth and viral gene expression in human papillomavirus type 16-containing cancer cell lines treated with progesterone.

This study explores interactions between high-risk human papillomavirus type 16 (HPV-16) and the female sex hormone progesterone in the growth of tumor cells and viral oncogene expression. For both the cervical cancer cell line CaSki containing integrated HPV-16 DNA and the laryngeal carcinoma cell line HEp-2 transfected with HPV-16 DNA, prolonged progesterone treatment enhances their colony formation efficiency both on plastic surface and in soft agar. In contrast, progesterone has no effect on the HPV-negative cervical cancer cell line C-33A or the untransfected HEp-2 parental cells. Progesterone increases HPV-16 E6/E7 oncogene transcription in both HPV-16-containing cell lines. A detectable increase requires at least 3 days of treatment, and this delayed response may be due, at least in part, to increased stability of viral transcripts as determined by actinomycin D treatment. The progesterone antagonist RU 486 and nuclease-resistant oligomers containing HPV-16 progesterone response element are able to abrogate the enhancement by progesterone on cell growth and E6/E7 gene transcription. Taken together, these results support the notion that progesterone can be a cofactor in HPV-related malignancies.

Carcinoma, Squamous Cell↗

PubMed1998

Endothelial cell death, angiogenesis, and microvascular function after castration in an androgen-dependent tumor: role of vascular endothelial growth factor.

The sequence of events that leads to tumor vessel regression and the functional characteristics of these vessels during hormone-ablation therapy are not known. This is because of the lack of an appropriate animal model and monitoring technology. By using in vivo microscopy and in situ molecular analysis of the androgen-dependent Shionogi carcinoma grown in severe combined immunodeficient mice, we show that castration of these mice leads to tumor regression and a concomitant decrease in vascular endothelial growth factor (VEGF) expression. Androgen withdrawal is known to induce apoptosis in Shionogi tumor cells. Surprisingly, tumor endothelial cells begin to undergo apoptosis before neoplastic cells, and rarefaction of tumor vessels precedes the decrease in tumor size. The regressing vessels begin to exhibit normal phenotype, i.e., lower diameter, tortuosity, vascular permeability, and leukocyte adhesion. Two weeks after castration, a second wave of angiogenesis and tumor growth begins with a concomitant increase in VEGF expression. Because human tumors often relapse following hormone-ablation therapy, our data suggest that these patients may benefit from combined anti-VEGF therapy.

Androgens↗

PubMed1998

Regulation of transport pathways in tumor vessels: role of tumor type and microenvironment.

Novel anti-neoplastic agents such as gene targeting vectors and encapsulated carriers are quite large (approximately 100-300 nm in diameter). An understanding of the functional size and physiological regulation of transvascular pathways is necessary to optimize delivery of these agents. Here we analyze the functional limits of transvascular transport and its modulation by the microenvironment. One human and five murine tumors including mammary and colorectal carcinomas, hepatoma, glioma, and sarcoma were implanted in the dorsal skin-fold chamber or cranial window, and the pore cutoff size, a functional measure of transvascular gap size, was determined. The microenvironment was modulated: (i) spatially, by growing tumors in subcutaneous or cranial locations and (ii) temporally, by inducing vascular regression in hormone-dependent tumors. Tumors grown subcutaneously exhibited a characteristic pore cutoff size ranging from 200 nm to 1.2 microm. This pore cutoff size was reduced in tumors grown in the cranium or in regressing tumors after hormone withdrawal. Vessels induced in basic fibroblast growth factor-containing gels had a pore cutoff size of 200 nm. Albumin permeability was independent of pore cutoff size. These results have three major implications for the delivery of therapeutic agents: (i) delivery may be less efficient in cranial tumors than in subcutaneous tumors, (ii) delivery may be reduced during tumor regression induced by hormonal ablation, and (iii) permeability to a molecule is independent of pore cutoff size as long as the diameter of the molecule is much less than the pore diameter.

Adenocarcinoma↗

PubMed1998

Transvascular drug delivery in solid tumors.

The microvessel wall is a barrier for the delivery of various therapeutic agents to tumor cells. Tumor microvessels are, in general, more permeable to macromolecules than normal vessels. The hyperpermeability is presumably due to the existence of large pore structures in the vessel wall, induced by various cytokines. The cutoff pore size is tumor dependent, as determined by transport studies of nanoparticles. The vascular permeability is heterogeneous in tumors and dependent on physicochemical properties of molecules as well as the ultrastructure of the vessel wall. The ultrastructure is dynamic and can be modulated by the tumor microenvironment. The microenvironment itself can be altered by the transvascular transport because the transport may facilitate angiogenesis, reduce blood flow, and induce interstitial hypertension in tumors. Future studies of transport need to address mechanisms of the barrier formation and emphasize development of novel strategies for circumventing or exploiting the vascular barrier.

Animals↗