Longitudinal optical monitoring of blood flow in breast tumors during neoadjuvant chemotherapy.

We measure tissue blood flow markers in breast tumors during neoadjuvant chemotherapy and investigate their correlation to pathologic complete response in a pilot longitudinal patient study (n = 4). Tumor blood flow is quantified optically by diffuse correlation spectroscopy (DCS), and tissue optical properties, blood oxygen saturation, and total hemoglobin concentration are derived from concurrent diffuse optical spectroscopic imaging (DOSI). The study represents the first longitudinal DCS measurement of neoadjuvant chemotherapy in humans over the entire course of treatment; it therefore offers a first correlation between DCS flow indices and pathologic complete response. The use of absolute optical properties measured by DOSI facilitates significant improvement of DCS blood flow calculation, which typically assumes optical properties based on literature values. Additionally, the combination of the DCS blood flow index and the tissue oxygen saturation from DOSI permits investigation of tissue oxygen metabolism. Pilot results from four patients suggest that lower blood flow in the lesion-bearing breast is correlated with pathologic complete response. Both absolute lesion blood flow and lesion flow relative to the contralateral breast exhibit potential for characterization of pathological response. This initial demonstration of the combined optical approach for chemotherapy monitoring provides incentive for more comprehensive studies in the future and can help power those investigations.

Imaging the redox states of human breast cancer core biopsies.

Currently, the gold standard to establish benign vs. malignant breast tissue diagnosis requires an invasive biopsy followed by tissue fixation for subsequent histopathological examination. This process takes at least 24 h resulting in tissues that are less suitable for molecular, functional, or metabolic analysis. We have recently conducted redox scanning (cryogenic NADH/flavoprotein fluorescence imaging) on snap-frozen breast tissue biopsy samples obtained from human breast cancer patients at the time of their breast cancer surgery. The redox state was readily determined by the redox scanner at liquid nitrogen temperature with extraordinary sensitivity, giving oxidized flavoproteins (Fp) an up to tenfold discrimination of cancer to non-cancer of breast in our preliminary data. Our finding suggests that the identified metabolic parameters could discriminate between cancer and non-cancer breast tissues without subjecting tissues to fixatives. The remainder of the frozen tissue is available for additional analysis such as molecular analysis and conventional histopathology. We propose that this novel redox scanning procedure may assist in tissue diagnosis in ex vivo tissues.

A mammographic dilemma: calcification or haemosiderin as a cause of opacities? Validation of a new digital diagnostic tool.

Core biopsies of an area of microcalcification demonstrated large collections of macrophages containing haemosiderin, with evidence of minimal microcalcification on H&E staining. Algorithms were developed that were capable of differentiating with high accuracy those signs due to calcification, using quantitative measurements such as the apparent volume composition of calcium. Using the linear attenuation coefficients of calcification and assuming an ellipsoid model for the 3-dimensional shape of calcification, we computed the relative calcification volume for each region of interest. The difference in the linear attenuation coefficients of iron and calcification allowed the two to be differentiated on a mammogram based on this measure of relative calcification volume.

GSTP1 CpG island DNA hypermethylation in hepatocellular carcinomas.

Glutathione S-transferases, enzymes that defend cells against damage mediated by oxidant and electrophilic carcinogens, may be critical determinants of cancer pathogenesis. We report here that the pathogenesis of hepatocellular carcinoma (HCC), one of the most common cancers in the world, frequently involves an accumulation of somatic DNA methylation changes at GSTP1, the gene encoding the pi-class glutathione S-transferase. For our study, Hep3B HCC cells and a cohort of 20 HCC tissue specimens were subjected to analysis for GSTP1 expression and for somatic GSTP1 alterations. GSTP1 DNA hypermethylation in HCC DNA was assessed by Southern blot analysis, via a polymerase chain reaction (PCR) assay, and by using a genomic sequencing approach. Hep3B HCC cells failed to express GSTP1 mRNA or GSTP1 polypeptides. Similarly, HCC cells in 19 of 20 HCC cases were devoid of GSTP1 polypeptides. By Southern blot analysis, DNA from Hep3B HCC cells displayed abnormal GSTP1 hypermethylation. Treatment of Hep3B HCC cells in vitro with the DNA methyltransferase inhibitor 5-aza-deoxycytidine both reversed GSTP1 DNA hypermethylation and restored GSTP1 expression. Using a PCR assay, somatic GSTP1 DNA hypermethylation was also detected in HCC DNA from 17 of 20 HCC cases. Genomic sequencing analyses, undertaken to map 5-methyldeoxycytidine nucleotides located at the GSTP1 transcriptional regulatory region, frequently detected somatic DNA hypermethylation near the gene promoter in HCC DNA. The data indicate that GSTP1 DNA hypermethylation changes appear frequently in human HCC. In addition, the data raise the possibility that somatic GSTP1 inactivation, via hypermethylation, may contribute to the pathogenesis of HCC.

The catalytic mechanism of Fpg protein. Evidence for a Schiff base intermediate and amino terminus localization of the catalytic site.

Our recent structure-activity analysis of Fpg protein of Escherichia coli, using oligodeoxynucleotides containing various 8-oxopurine derivatives, has allowed us to postulate an enzyme mechanism involving protonation of 8-oxoguanine at O-6 and nucleophilic attack of the deoxyribose moiety at C-1' leading to the formation of an enzyme-substrate Schiff base intermediate (Tchou, J., Bodepudi, V., Shibutani, S., Antoshechkin, I., Miller, J., Grollman, A. P., and Johnson, F. (1994) J. Biol. Chem. 269, 15318-15324). In this paper, sodium cyanoborohydride has been used to convert the transient intermediate to a covalent enzyme-DNA complex. The location of the active site of Fpg protein is further delineated using two approaches. 1) A radiolabeled DNA substrate is used to tag the active site of Fpg protein, using sodium cyanoborohydride. The active site is mapped to the first 73 amino acid residue fragment by cyanogen bromide cleavage analysis. 2) A maltose-binding protein fusion system is used to generate amino-terminal modifications of Fpg protein to explore the role of the amino-terminal region in DNA binding and catalysis. Results support the conclusion that the active site of Fpg protein is located at or near the amino terminus. Thus, Fpg protein may act in a similar fashion as T4 endonuclease V, a DNA repair enzyme that uses its amino-terminal alpha-amino group of threonine to carry out catalysis via Schiff base formation (Dodson et al., 1993).

Substrate specificity of Fpg protein. Recognition and cleavage of oxidatively damaged DNA.

The 8-oxoguanine-DNA glycosylase of Escherichia coli, also known as formamidopyrimidine-DNA glycosylase (Fpg protein), has N-glycosylase and AP-lyase activities. This enzyme repairs oxidative DNA damage by efficiently removing formamidopyrimidine lesions and 8-oxoguanine residues from DNA. Defined oligodeoxynucleotides containing various 8-oxopurines were used to examine the substrate specificity of Fpg protein and to establish the role of functional groups in DNA on damage recognition and catalysis. Binding affinities of Fpg protein were established for duplex oligodeoxynucleotides containing 8-oxo-2'-deoxyguanine, 8-oxo-2'-deoxyadenine, 8-oxo-2'-deoxynebularine, 8-oxo-2'-deoxyinosine, abasic sites, and a ring-open adduct of C8-aminofluorene guanine. The C8 keto group of 8-oxodG:dC presents in the major groove and is correlated with tight binding (Kd = 8.9 nM). Binding is much weaker when the C8 keto functional group is in the minor groove, as in 8-oxodG:dA (Kd = 340 nM). Km and Vmax were determined for the cleavage reaction. Specificity constants (Kcat/Km) are consistently higher for oligodeoxynucleotide duplexes containing 8-oxopurines with C6 and C8 keto groups, as in 8-oxodG:dC and 8-oxodI:dC, where Kcat/Km are 9.3 and 18 min-1 nM x 10(-3), respectively. 8-oxodN:dC lacks the C6 keto group; the specificity constant is 0.024 min-1 nM x 10(-3). Taken together, our data suggest that the C8 keto group of 8-oxodeoxyguanine and the carbonyl moiety of formamidopyrimidine enable Fpg protein to recognize and bind duplex DNA containing these modified bases. An enzyme-catalyzed reaction involving the C6 keto group of the substrate leads to removal of these lesions. A mechanism involving protonation at O-6 of 8-oxoguanine is proposed to account for the N-glycosylase activity of this enzyme.

Function of the zinc finger in Escherichia coli Fpg protein.

Fpg protein of Escherichia coli cleaves duplex DNA containing the oxidatively damaged base 8-oxo-7,8-dihydroguanine (Tchou, J., Kasai, H., Shibutani, S., Chung, M.-H., Laval, J., Grollman, A. P., and Nishimura, S. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4690-4694). This DNA repair enzyme contains one zinc atom/protein molecule (Boiteux, S., O'Connor, T. R., Lederer, F., Gougette, A., and Laval, J. (1990) J. Biol. Chem. 265, 3916-3922); its N-glycosylase and apurinic/apyrimidinic lyase activities are physically associated. Amino acid sequence analysis reveals a putative single zinc finger motif of the CC/CC type located near the carboxyl terminus. A gel mobility shift assay was used to assay binding of Fpg protein to a noncleavable substrate analog, namely an oligodeoxynucleotide duplex containing a single tetrahydrofuran residue. High resolution hydroxyl radical DNA footprinting showed protection centered around the tetrahydrofuran residue. No footprint was observed on the complementary strand. To establish the role of COOH-terminal zinc finger in DNA binding and/or DNA cleavage, amino acid substitutions and an amber mutation were introduced at Cys-244 (C244S, C244H, C244A, and C244amber). In addition, a double amino acid substitution was generated at Cys-244 and Cys-247 (C244S/C247S). These mutant Fpg proteins lack DNA binding or cleavage activity, as tested in crude lysates of Escherichia coli. Wild type Fpg protein contains one zinc/protein molecule, whereas the mutant Fpg protein (C244S/C247S) lacks zinc, as measured by atomic absorption spectroscopy. This mutation did not significantly alter secondary structure, as assessed by circular dichroism spectroscopy. Our results suggest that Fpg protein utilizes its single COOH-terminal zinc finger motif in DNA binding.

A repair system for 8-oxo-7,8-dihydrodeoxyguanine.

Active oxygen species can damage DNA and may play a role in aging and carcinogenesis. We have tested MutY glycosylase for activity on undamaged mispairs as well as mispairs formed with the oxidatively damaged substrates, 8-oxo-7,8-dihydrodeoxyguanine (GO) or 8-oxo-7,8-dihydrodeoxyadenine (AO). MutY acts as a glycosylase on four of the heteroduplexes tested, A/G, A/GO, A/C, and A/AO, removing the undamaged adenine from each substrate. Genetic data suggest that the primary substrate for MutY glycosylase in vivo is the A/GO mispair. We present biochemical evidence demonstrating that MutY glycosylase is an important part of a repair system that includes the MutM and MutT proteins. The GO repair system is dedicated to the repair of the oxidatively damaged guanine and the mutations it can induce.

8-oxoguanine (8-hydroxyguanine) DNA glycosylase and its substrate specificity.

Substrate specificities of FPG protein (also known as formamidopyrimidine DNA glycosylase) and 8-hydroxyguanine endonuclease were compared by using defined duplex oligodeoxynucleotides containing single residues of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxodA), and 2,6-diamino-4-hydroxy-5-(N-methyl)formamidopyrimidine (Me-Fapy). Duplexes containing 8-oxodG positioned opposite dC, dG, or dT were cleaved, whereas single-stranded DNA and duplexes containing 8-oxodG.dA or 8-oxodA positioned opposite any of the four DNA bases were relatively resistant. Both enzymes cut duplexes containing 8-oxoG.dC 3' and 5' to the modified base but failed to cleave duplex DNA containing synthetic abasic sites, mismatches containing dG, or unmodified DNA. 8-Oxoguanine, identified by HPLC-electrochemical detection techniques, was released during the enzymatic reaction. Apparent Km values for FPG protein acting on duplex substrates containing a single Me-Fapy or 8-oxodG residue positioned opposite dC were 41 and 8 nM, respectively, and those for 8-hydroxyguanine endonuclease were 30 and 13 nM, respectively. Comparison of the properties of the two enzyme activities suggest that they are identical. In view of the widespread distribution of 8-oxodG in cellular DNA, the demonstrated miscoding and mutagenic properties of this lesion, and the existence of a bacterial gene coding for FPG protein, we propose that 8-oxodG DNA is the primary physiological substrate for a constituent glycosylase found in bacteria and mammalian cells.