Patients' understanding of how genotype variation affects benefits of tamoxifen therapy for breast cancer.

BACKGROUND: CYP2D6 is a critical enzyme in the metabolism of tamoxifen and potentially a key determinant in breast cancer outcomes. Our study examined patients' beliefs about how the CYP2D6 genotype would affect their prognoses. METHODS: Women enrolled in a pharmacogenomic clinical trial and on tamoxifen for prevention or treatment of breast cancer underwent CYP2D6 genotyping (EM = extensive, IM = intermediate, PM = poor metabolizing alleles). The informed consent said that the purpose of the trial was to examine effects of dose adjustment based on genotype, but that clinical benefits were uncertain. Our embedded sub-study surveyed 320 patients prior to receiving their genotypes. We experimentally manipulated 6 vignettes to describe hypothetical tamoxifen treatment (no or yes) and hypothetical genotype (EM, IM or PM). For each vignette, women gave their perceived recurrence risk (RR; 0-100%). RESULTS: Women believed that genotype would not affect their RR if they did not take tamoxifen (p = 0.06). However, women believed that if prescribed tamoxifen, genotype would affect their RR (22% if EM, 30% if IM and 40% if PM, p < 0.001). CONCLUSION: Women believed that extensive tamoxifen metabolizers had better prognoses, despite study materials stating uncertainty about any benefit. The rapidly changing nature of genomic science calls for caution when communicating clinical utility.

Who gets genomic testing for breast cancer recurrence risk?

BACKGROUND/AIMS: Our study examined whether patient characteristics, beliefs and decision-making styles were associated with uptake of genomic testing for breast cancer recurrence risk. METHODS: Participants were 132 early-stage breast cancer patients eligible for the Oncotype DX genomic test. We interviewed patients in 2009-2010 and obtained information from medical charts. RESULTS: Half of the women eligible for genomic testing for breast cancer recurrence risk received it. The most common reason for not getting the test was that women's physicians did not offer it (80%). Test recipients were more likely to be unsure about receiving chemotherapy treatment compared to women who did not receive the test (p < 0.05). Women who received the test had less advanced disease pathologies, recalled a lower objective recurrence risk, perceived lower recurrence risk, and were slightly younger (all p < 0.05). Most women who described their decision-making style as active received the test (75%), whereas few women who described their style as passive received the test (12%) (p < 0.01). CONCLUSION: In the university clinic we studied, genomic testing appeared to be more common among patients who may benefit most from the information provided by results, but confirmation in larger studies is needed.

Effect of organophosphorus hydrolysing enzymes on obidoxime-induced reactivation of organophosphate-inhibited human acetylcholinesterase.

The reactivation of organophosphate (OP)-inhibited acetylcholinesterase (AChE) by oximes results inevitably in the formation of highly reactive phosphyloximes (POX), which may re-inhibit the enzyme. An impairment of net reactivation by stable POX was found with 4-pyridinium aldoximes, e.g. obidoxime, and a variety of OP compounds. In this study the effect of organophosphorus hydrolase (OPH), organophosphorus acid anhydrolase (OPAA) and diisopropylfluorophosphatase (DFPase) on obidoxime-induced reactivation of human acetylcholinesterase (AChE) inhibited by different OPs was investigated. Reactivation of paraoxon-, sarin-, soman- and VX-inhibited AChE by obidoxime was impaired by POX-induced re-inhibition whereas no deviation of pseudo first-order kinetics was observed with tabun, cyclosarin and VR. OPH prevented (paraoxon) or markedly reduced the POX-induced re-inhibition (VX, sarin, soman), whereas OPAA and DFPase were without effect. Additional experiments with sarin-inhibited AChE indicate that the POX hydrolysis by OPH was concentration-dependent. The activity of OP-inhibited AChE was not affected by OPH in the absence of obidoxime. In conclusion, OPH may be a valuable contribution to the therapeutic regimen against OP poisoning by accelerating the degradation of both the parent compound, OP, and the reaction product, POX.

Deficiency of human BRCA2 leads to impaired homologous recombination but maintains normal nonhomologous end joining.

Carriers of BRCA2 germline mutations are at high risk to develop early-onset breast cancer. The underlying mechanisms of how BRCA2 inactivation predisposes to malignant transformation have not been established. Here, we provide direct functional evidence that human BRCA2 promotes homologous recombination (HR), which comprises one major pathway of DNA double-strand break repair. We found that up-regulated HR after transfection of wild-type (wt) BRCA2 into a human tumor line with mutant BRCA2 was linked to increased radioresistance. In addition, BRCA2-mediated enhancement of HR depended on the interaction with Rad51. In contrast to the tumor suppressor BRCA1, which is involved in multiple DNA repair pathways, BRCA2 status had no impact on the other principal double-strand break repair pathway, nonhomologous end joining. Thus, there exists a specific regulation of HR by BRCA2, which may function to maintain genomic integrity and suppress tumor development in proliferating cells.

Stereochemical specificity of organophosphorus acid anhydrolase toward p-nitrophenyl analogs of soman and sarin.

Organophosphorus acid anhydrolase (OPAA) catalyzes the hydrolysis of p-nitrophenyl analogs of the organophosphonate nerve agents, sarin and soman. The enzyme is stereoselective toward the chiral phosphorus center by displaying a preference for the R(P)-configuration of these analogs. OPAA also exhibits an additional preference for the stereochemical configuration at the chiral carbon center of the soman analog. The preferred configuration of the chiral carbon center is dependent upon the configuration at the phosphorus center. The enzyme displays a two- to four-fold preference for the R(P)-enantiomer of the sarin analog. The k(cat)/K(m) of the R(P)-enantiomer is 250 M(-1) s(-1), while that of the S(P)-enantiomer is 110 M(-1) s(-1). The order of preference for the stereoisomers of the soman analog is R(P)S(C) > R(P)R(C) > S(P)R(C) > S(P)S(C). The k(cat)/K(m) values are 36,300 M(-1)s(-1), 1250 M(-1) s(-1), 80 M(-1) s(-1) and 5 M(-1) s(-1), respectively. The R(P)S(C)-isomer of the soman analog is therefore preferred by a factor of 7000 over the S(P)S(C)-isomer.

Interaction between organophosphorous hydrolase and paraoxon studied by surface chemistry in situ at air-water interface.

Subphase conditions have been optimized to obtain stable organophosphorous hydrolase (OPH-EC 3.1.8.1) as Langmuir films. The Langmuir film was characterized by surface pressure and surface potential-area isotherms and UV-Vis spectroscopy in situ. The interaction of an organophosphorous compound, namely Paraoxon, with the OPH film was investigated for various surface pressures. The stability of the monolayer and the evidence of the enzyme activity at air-water interface support the use of enzyme LB films as biosensor.

Long circulating liposomes encapsulating organophosphorus acid anhydrolase in diisopropylfluorophosphate antagonism.

These studies are focused on antagonizing organophosphorous (OP) intoxications by a new conceptual approach using recombinant enzymes encapsulated within sterically stabilized liposomes to enhance diisopropylfluorophosphate (DFP) degradation. The OP hydrolyzing enzyme, organophosphorous acid anhydrolase (OPAA), encapsulated within the liposomes, was employed either alone or in combination with pralidoxime (2-PAM) and/or atropine. The recombinant OPAA enzyme, from the ALTEROMONAS: strain JD6, has high substrate specificity toward a wide range of OP compounds, e.g., DFP, soman, and sarin. The rate of DFP hydrolysis by liposomes containing OPAA (SL)* was measured by determining the changes in fluoride-ion concentration using a fluoride ion-selective electrode. This enzyme carrier system serves as a biodegradable protective environment for the OP-metabolizing enzyme (OPAA), resulting in an enhanced antidotal protection against the lethal effects of DFP. Free OPAA alone showed some antidotal protection; however, the protection with 2-PAM and/or atropine was greatly enhanced when combined with (SL)*.

In vitro studies on sterically stabilized liposomes (SL) as enzyme carriers in organophosphorus (OP) antagonism.

This study describes a new approach for organophosphorous (OP) antidotal treatment by encapsulating an OP hydrolyzing enzyme, OPA anhydrolase (OPAA), within sterically stabilized liposomes. The recombinant OPAA enzyme was derived from Alteromonas strain JD6. It has broad substrate specificity to a wide range of OP compounds: DFP and the nerve agents, soman and sarin. Liposomes encapsulating OPAA (SL)* were made by mechanical dispersion method. Hydrolysis of DFP by (SL)* was measured by following an increase of fluoride ion concentration using a fluoride ion selective electrode. OPAA entrapped in the carrier liposomes rapidly hydrolyze DFP, with the rate of DFP hydrolysis directly proportional to the amount of (SL)* added to the solution. Liposomal carriers containing no enzyme did not hydrolyze DFP. The reaction was linear and the rate of hydrolysis was first order in the substrate. This enzyme carrier system serves as a biodegradable protective environment for the recombinant OP-metabolizing enzyme, OPAA, resulting in prolongation of enzymatic concentration in the body. These studies suggest that the protection of OP intoxication can be strikingly enhanced by adding OPAA encapsulated within (SL)* to pralidoxime and atropine.

Substrate and stereochemical specificity of the organophosphorus acid anhydrolase from Alteromonas sp. JD6.5 toward p-nitrophenyl phosphotriesters.

The enzyme OPAA hydrolyzes p-nitrophenyl phosphotriesters bearing substituents at the phosphorus center ranging in size from methyl to phenyl. The enzyme exhibits stereoselectivity toward the hydrolysis of chiral substrates with a preference for the Sp enantiomer.

Alteromonas prolidase for organophosphorus G-agent decontamination.

Enzymes catalyzing the hydrolysis of highly toxic organophosphorus compounds (OPs) are classified as organophosphorus acid anhydrolases (OPAA; EC 3.1.8.2). Recently, the genes encoding OPAA from two species of Alteromonas were cloned and sequenced. Sequence and biochemical analyses of the cloned genes and enzymes have established Alteromonas OPAAs to be prolidases (E.C. 3.4.13.9), a type of dipeptidase hydrolyzing dipeptides with a prolyl residue in the carboxyl-terminal position (X-Pro). Alteromonas prolidases hydrolyze a broad range of G-type chemical warfare (CW) nerve agents. Efforts to over-produce a prolidase from A. sp.JD6.5 with the goal of developing strategies for long-term storage and decontamination have been successfully achieved. Large-scale production of this G-agent degrading enzyme is now feasible with the availability of an over-producing recombinant cell line. Use of this enzyme for development of a safe and non-corrosive decontamination system is discussed.

The ability of p53 to activate downstream genes p21(WAF1/cip1) and MDM2, and cell cycle arrest following DNA damage is delayed and attenuated in scid cells deficient in the DNA-dependent protein kinase.

scid mouse embryonic fibroblasts are deficient in DNA-dependent protein kinase activity due to a mutation in the C-terminal domain of the catalytic subunit (DNA-PKcs). When exposed to ionizing radiation, the increase in levels of p53 was the same as in normal mouse embryonic fibroblasts. However, the rise in p21(WAF1/cip1) and mdm2 was found to be delayed and attenuated, which correlated in time with delayed onset of G1/S arrest by flow cytometric analysis. The p53-dependent G1 checkpoint was not eliminated: inactivation of p53 by the E6 protein in scid cells resulted in the complete loss of detectable G1/S arrest after DNA damage. Immunofluorescence analysis of normal cells revealed p53 to be localized predominantly within the cytoplasm prior to irradiation and then translocate to the nucleus after irradiation. In contrast, scid cells show abnormal accumulation of p53 in the nucleus independent of irradiation, which was confirmed by immunoblot analysis of nuclear lysates. Taken together, these data suggest that loss of DNA-PK activity appears to attenuate the kinetics of p53 to activate downstream genes, implying that DNA-PK plays a role in post-translational modification of p53, without affecting the increase in levels of p53 in response to DNA damage.

phnE and glpT genes enhance utilization of organophosphates in Escherichia coli K-12.

Wild-type Escherichia coli K-12 strain JA221 grows poorly on low concentrations (< or = 1 mM) of diisopropyl fluorophosphate and its hydrolysis product, diisopropyl phosphate (DIPP), as sole phosphorus sources. Spontaneous organophosphate utilization (OPU) mutants were isolated that efficiently utilized these alternate sources of phosphate. A genomic library was constructed from one such OPU mutant, and two genes were isolated that conferred the OPU phenotype to strain JA221 upon transformation. These genes were identified as phnE and glpT. The original OPU mutation represented phnE gene activation and corresponded to the same 8-bp unit deletion from the cryptic wild-type E. coli K-12 phnE gene that has been shown previously to result in phnE activation. In comparison, sequence analysis revealed that the observed OPU phenotype conferred by the glpT gene was not the result of a mutation. PCR clones of glpT from both the mutant and the wild type were found to confer the OPU phenotype to JA221 when they were present on the high-copy-number pUC19 plasmid but not when they were present on the low-copy-number pWSK29 plasmid. This suggests that the OPU phenotype associated with the glpT gene is the result of amplification and overproduction of the glpT gene product. Both the active phnE and multicopy glpT genes facilitated effective metabolism of low concentrations of DIPP, whereas only the active phnE gene could confer the ability to break down a chromogenic substrate, 5-bromo-4-chloro-3-indoxyl phosphate-p-toluidine (X-Pi). This result indicates that in E. coli, X-Pi is transported exclusively by the Phn system, whereas DIPP (or its metabolite) may be transported by both Phn and Glp systems.

Inactivation of p53 results in high rates of homologous recombination.

Using a plasmid substrate which integrates into the genome, we determined that the rate of homologous recombination was suppressed by p53. Human tumor cell lines, mutant or null for p53 had recombination rates 10000-times greater than primary fibroblasts. When isogenic cell pairs from tumor cells or primary fibroblasts were compared, differing only in one genetic change which inactivated p53, the recombination rate increased > 100-fold. Functional inactivation of p53 by dominant mutant p53, by large T antigen of SV40 virus, by E6 protein of human papilloma virus, or by genetic deletion led to the same result. Our results suggest that p53 suppresses spontaneous homologous recombination, and that p53 is not required for recombination to proceed. The mechanism of recombination suppression may be related to the reported association of p53 with Rad 51, but the functional consequences of this association are not yet established. It is suggested that suppression of homologous recombination is the means by which p53 maintains genetic stability.

Nucleotide sequence of a gene encoding an organophosphorus nerve agent degrading enzyme from Alteromonas haloplanktis.

Organophosphorus acid anhydrolases (OPAA) catalyzing the hydrolysis of a variety of toxic organophosphorus cholinesterase inhibitors offer potential for decontamination of G-type nerve agents and pesticides. The gene (opa) encoding an OPAA was cloned from the chromosomal DNA of Alteromonas haloplanktis ATCC 23821. The nucleotide sequence of the 1.7 -kb DNA fragment contained the opa gene (1.3 kb) and its flanking region. We report structural and functional similarity of OPAAs from A. haloplanktis and Alteromonas sp JD6.5 with the enzyme prolidase that hydrolyzes dipeptides with a prolyl residue in the carboxyl-terminal position. These results corroborate the earlier conclusion that the OPAA is a type of X-Pro dipeptidase, and that X-Pro could be the native substrate for such an enzyme in Alteromonas cells.

Enzymatic hydrolysis of Russian-VX by organophosphorus hydrolase.

The Russian-VX (R-VX) is the principle V-type nerve agent in the chemical warfare (CW) arsenal of the Former Soviet Union. We here report the enzymatic hydrolysis of the P-S bond of Russian-VX by organophosphorus hydrolase (OPH) from Pseudomonas diminuta. While the Michaelis constant, K(m) for R-VX (474 microM), was similar to that for VX (434 microM), the Vmax for R-VX (2.1 mumoles/mg/min) was about four-fold higher compared to that for VX (0.56 mumoles/mg/min). A 50% inhibition in the rate of the enzymatic hydrolysis of R-VX was observed in the presence of 0.5% ethanol, isoamyl-alcohol, or isopropanol. The presence of acetonitrile, diethylene glycol, or methanol had marginal effects. These results comprise the first demonstration of enzymatic detoxification of R-VX.

p53-null cells are more sensitive to ultraviolet light only in the presence of caffeine.

We have shown previously that p53(-/-) fibroblasts show greater sensitization by caffeine to the lethal effects of ionizing radiation compared with p53(+/+) cells. Recently published data have suggested a possible role of p53 in nucleotide excision repair: an association of p53 and xeroderma pigmentosum group B protein and a greater sensitivity to cisplatin of RKO cells transfected with the E6 protein of human papilloma virus (inactivating p53). We show that p53(+/+) and p53(-/-) cells have equal sensitivity to germicidal UV light (as with ionizing radiation). However, the introduction of 2 mM caffeine led to a sensitization enhancement ratio (at 10% survival) of 1.8 in p53(-/-) cells, but only 1.3 in wild-type (p53+/+) cells. Lower doses of caffeine had less effect, and 0.1 mM caffeine resulted in no detectable sensitization of either cell type to UV light in contrast to X-rays. The differential sensitivity of p53(-/-) cells to X-rays and caffeine was thought to be due to override of the G2-M block to cell cycle progression. In response to UV light, cells accumulate in S phase, and the magnitude of S-phase accumulation was observed to be greater in p53(-/-) cells. Caffeine had little effect on the cell cycle distribution in p53(+/+) cells. However, for p53(-/-) cells, a greater proportion were in S phase after treatment with caffeine, and a complete loss of S-phase delay was observed after UV irradiation. In conclusion, the role of p53 in nucleotide excision repair appears to be of little significance for cell survival. Greater sensitization of p53(-/-) cells to caffeine could be mediated via override of S-phase delay.

High frequency and error-prone DNA recombination in ataxia telangiectasia cell lines.

The only specific DNA repair defect found in ataxia telangiectasia (A-T) cells is mis-repair of cleaved DNA. In this report we measured DNA recombination, given its role in DNA repair and genetic instability. Using plasmids containing selectable reporter genes, we found a higher frequency of both chromosomal recombination (>100 times) and extra-chromosomal recombination (27 times) in SV40-transformed A-T cell lines compared with in an SV40-transformed normal fibroblast cell line. Southern analysis of single A-T colonies exhibiting post-integration recombination revealed that 24/27 had undergone aberrant rearrangements; recombination in normal fibroblast colonies was achieved by gene conversion in 8/11 clones and 10/11 clones showed unchanged copies of the plasmid. Using co-transfection of two integrating plasmids, each containing a separate deletion in the xgprt reporter gene, the 27 times difference in extra-chromosomal recombination was found when the plasmids were cleaved at a distance from the reporter gene. When the plasmids were cleaved within the reporter gene, the co-transfection frequency was reduced in A-T, but was increased in normal cells. We conclude that A-T cell lines have not only a high frequency chromosomal and extra-chromosomal recombination, but also exhibit error-prone recombination of cleaved DNA.

Differential sensitivity of p53(-) and p53(+) cells to caffeine-induced radiosensitization and override of G2 delay.

Most drug discovery efforts have focused on finding new DNA-damaging agents to kill tumor cells preferentially. An alternative approach is to find ways to increase tumor-specific killing by modifying tumor-specific responses to that damage. In this report, we ask whether cells lacking the G1-S arrest in response to X-rays are more sensitive to X-ray damage when treated with agents that override G2-M arrest. Mouse embryonic fibroblasts genetically matched to be (+) or (-) p53 and rat embryonic fibroblasts (+) or (-) for wild-type p53 function were irradiated with and without caffeine, a known checkpoint inhibitor. At low doses (500 microM), caffeine caused selective radiosensitization in the p53(-) cells. At this low dose (where no effect was seen in p53(+) cells), the p53(-) cells showed a 50% reduction in the size of the G2-M arrest. At higher doses (2 mM caffeine), where sensitization was seen in both p53(+) and p53(-) cells, the radiosensitization and the G2-M override were more pronounced in the p53(-) cells. The greater caffeine-induced radiosensitization in p53(-) cells suggests that p53, already shown to control the G1-S checkpoint, may also influence aspects of G2-M arrest. These data indicate an opportunity for therapeutic gain by combining DNA-damaging agents with compounds that disrupt G2-M arrest in tumors lacking functional p53.

Screening of halophilic bacteria and Alteromonas species for organophosphorus hydrolyzing enzyme activity.

Previously, a G-type nerve agent degrading enzyme activity was found in a halophilic bacterial isolate designated JD6.5. This organism was tentatively identified as an unknown species of the genus Alteromonas. In order to determine whether this type of enzyme activity was common in other species of Alteromonas, a screening program was initiated. A number of Alteromonas species and five halophilic bacterial isolates were cultured and their crude cell extracts screened for hydrolytic activity against several organophosphorus chemical agents and other related compounds. The samples were also screened for cross-reactivity with a monoclonal antibody raised against the purified enzyme from JD6.5 and for hybridization with a DNA probe based on its N-terminal amino acid sequence A wide spectrum of activities and reactivities were seen, suggesting a significant heterogeneity between the functionally similar enzymes that are present in these bacterial species. Enzymes of the type described here have considerable potential for the decontamination and demilitarization of chemical warfare agents.