Predictive value of p53, mdm-2, p21, and mib-1 for chemotherapy response in advanced breast cancer.

p53 is a transcription factor that participates in cell cycle checkpoint processes and apoptosis. The protein product of the murine double minute gene 2 (mdm-2) plays a central role in the regulation of p53. In response to DNA-damaging agents, the wild-type p53-activated fragment 1 (WAF1 also known as p21) is an important downstream effector in the p53-specific growth arrest pathway. In breast cancer patients, it is unclear whether measuring p53, mdm-2, or p21 expression provides information on how patients will respond to chemotherapy. Mib-1 monoclonal antibody recognizes the proliferation-related antigen Ki-67. High tumor proliferation has previously been associated with response to chemotherapy. p53, mdm-2, p21, and mib-1 expression were assessed by immunohistochemical methods in primary tumors derived from 134 patients who took part in a randomized multicenter trial comparing docetaxel to sequential methotrexate and 5-fluorouracil (MF) in advanced breast cancer. Low mib-1 staining correlated with negative p53 staining (P = 0.001), and mdm-2 and p21 stainings correlated positively with each other (P < 0.001). p53, mdm-2, p21, and mib-1 expression were not significantly associated with response to chemotherapy, time to progression, or overall survival in the whole patient population or in the docetaxel group. However, in the MF group, a low mib expression (<25%) and a high mdm-2 expression (> or =10%) predicted a better response (P = 0.014 and P = 0.046, respectively) to treatment and a longer time to progression in both univariate and multivariate analyses. p53 staining status was not associated with response to treatment in either group. Interestingly, tumors with both negative mdm-2 and p21 expression, irrespective of p53 status, had a high response rate to docetaxel but no response to MF. Although highly preliminary, the findings suggest that different tumor biological factors may predict response to different chemotherapy regimens with distinct mechanisms of action. The results of our phenotype analysis also indicate that it is more likely that a panel of tumor biological factors instead of only one single factor may be needed for better prediction of chemotherapy response.

Characterization of small plasmids from Staphylococcus aureus.

Small molecular weight plasmids from Staphylococcus aureus were characterized with respect to size, restriction enzyme cleavage pattern and transforming capacity. The plasmids pS194 and pC194 which encode streptomycin and chloramphenicol resistance respectively contained 3.0 and 2.0 megadaltons of DNA as determined by zonal rate centrifugation and electron-microscopy. Both plasmids transformed S. aureus wigh high efficiency. Plasmid pC194 contained only one cleavage site for endonuclease HindIII and pS194 contained single cleavage sites for HindIII and EcoRI. A natural recombinant between these two plasmids, pSC194, shared the high transforming capacity of the parental plasmids and contained one EcoRI site and two HindIII sites. pSC194 DNA also transformed B. subtilis with high efficiency. The recombinant plasmid pSC194 may be used as an EcoRI vector for construction and propagation of hybrid DNA in S. aureus as shown in the following paper (Löfdahl et al., 1978).

A vector for recombinant DNA in Staphylococcus aureus.

Staphylococcal plasmids pS194 and pSC194 which confer streptomycin and streptomycin-chloramphenicol resistance respectively have been used as vectors for construction of recombinant DNA, since they each carry one single recipient site for endonuclease EcoRI. Hybrid DNA does not express streptomycin resistance, a marker which is present in both vectors, presumably because the marker gene is cleaved by EcoRI. A chloramphenicol marker present in pSC194 was used for positive hybrid selection. Hybrid plasmids generated by joining pSC194 with one or more of the four EcoRI fragments of the large (18.1-10(6) daltons) staphylococcal plasmid pI258 were constructed and permitted us to develop a physical map for pI258.

Prognostic value of quality of life scores for time to progression (TTP) and overall survival time (OS) in advanced breast cancer.

The purpose of the study was to investigate whether baseline quality of life (QoL) and changes in QoL scores from baseline are prognostic for time to progression (TTP) and/or overall survival (OS) in patients with advanced breast cancer receiving docetaxel (T) or sequential methotrexate and 5-fluorouracil (MF). QoL was assessed at baseline and before each treatment using the European Organization for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire (QLQ-C30). Survival curves and probabilities were estimated using the Kaplan-Meier technique. The Cox proportional hazards regression model was used for both the univariate and multivariate analyses to explore relationships between baseline QoL variables and TTP, as well as OS. In the univariate analysis, more severe pain and fatigue at baseline were predictive for a shorter OS; global QoL, physical functioning and appetite loss had a borderline significance (P=0.0130 for global QoL; P=0.0256 for physical functioning: P=0.0149 for appetite loss). World Health Organization (WHO) performance status was significantly predictive for OS. In the multivariate analysis, more severe pain at baseline was predictive for a shorter OS. In contrast, baseline QoL had no prognostic value for the duration of TTP. QoL change scores from baseline QoL predicted neither OS nor TTP. Our findings suggest that while QoL measurements are important in evaluating patients' QoL, they have no great importance in predicting primary clinical endpoints such as TTP or OS in advanced breast cancer patients.

C-erbB-2 expression does not predict response to docetaxel or sequential methotrexate and 5-fluorouracil in advanced breast cancer.

Breast cancer patients with c-erbB-2-positive tumours seem to benefit from anthracycline-based adjuvant chemotherapy. The predictive value of c-erbB-2 for taxane sensitivity is not yet clear. The purpose of this study was to assess whether c-erbB-2 expression is associated with clinical sensitivity to docetaxel (T) or sequential methotrexate and 5-fluorouracil (MF). A total of 283 patients with metastatic breast cancer were initially enrolled in a randomised multicentre trial comparing docetaxel with sequential MF in advanced breast cancer. Paraffin-embedded blocks of the primary tumour were available for 131 patients (46%). c-erbB-2 status was determined by immunohistochemistry using a polyclonal antibody to the c-erbB-2 protein. C-erbB-2 expression was scored in a semi-quantitative fashion using a 0 to 3+ scale. Staining scores 2+ or greater were considered positive. Response evaluation was performed according to World Health Organization (WHO) recommendations. Overall 54 (42%) patients had c-erbB-2-positive tumours. There was no association between treatment outcome and c-erbB-2 overexpression. The overall response rates (RR) (n=128) among c-erbB-2-negative and -positive patients were 35 and 44%, respectively (P=0.359). In the MF arm (n=62), the RR was somewhat higher in the c-erbB-2 overexpressors (33% versus 18%, P=0.18). In the docetaxel arm the RRs were very similar, regardless of the c-erbB-2 expression (53% versus 53%). While several studies have suggested a prognostic and putative predictive significance of c-erbB-2 overexpression in early breast cancer, the significance of c-erbB-2 expression as a predictive factor for response to various cytotoxic treatments in advanced breast cancer is still controversial. In this study, c-erbB-2 expression could not predict response to either MF or T. Thus, tumours over-expressing c-erbB-2 are not uniformly more sensitive to taxanes and c-erbB-2 expression cannot yet be applied clinically as a predictive factor for response in advanced breast cancer.

Docetaxel compared with sequential methotrexate and 5-fluorouracil in patients with advanced breast cancer after anthracycline failure: a randomised phase III study with crossover on progression by the Scandinavian Breast Group.

The aim of this study was to compare the efficacy and tolerability of docetaxel to methotrexate and 5-fluorouracil in advanced breast cancer after anthracycline failure. A randomised multicentre trial was conducted in 283 patients with advanced breast cancer who had failed previous anthracycline treatment. Docetaxel at a dose of 100 mg/m2 every 3 weeks (n = 143) was compared with sequential methotrexate and 5-fluorouracil (MF; n = 139) given at day 1 and 8 every 3 weeks at dosages of 200 mg/ m2 and 600 mg/m2, respectively. After progression, crossover to the alternative treatment group was recommended. There was a significantly higher overall response rate in the docetaxel 42% (CR 8% + PR 34%) than in the MF arm 21% (CR 3% + PR 18%) (P < 0.001). The median time to progression (TTP) was 6.3 months in the docetaxel arm and 3.0 months in the MF arm (P < 0.001). Docetaxel also had a significantly higher response rate of 27% following crossover compared with MF (12%). Significantly more side-effects (leucopenia, infections, neuropathy, oedema, asthenia, skin, nail changes, alopecia) were seen in the docetaxel than in the MF group. However, grade 3 and 4 side-effects were infrequent with both drugs, with the exception of fatigue, alopecia and infections. Median overall survival (OS) including crossover phase was 10.4 months in the docetaxel and 11.1 months in the MF arm (P = 0.79). Based on the response rate and the primary endpoint of TTP, docetaxel is superior to sequential methotrexate and 5-fluorouracil in advanced breast cancer after anthracycline failure.

Quality of life in patients with metastatic breast cancer receiving either docetaxel or sequential methotrexate and 5-fluorouracil. A multicentre randomised phase III trial by the Scandinavian breast group.

The purpose of this study was to evaluate the effects of two alternative chemotherapy regimes on the quality of life (QoL) of patients with advanced breast cancer. In a multicentre trial, 283 patients were randomised to receive either docetaxel (T) or sequential methotrexate and 5-fluorouracil (MF). QoL was assessed at baseline and before each treatment using the European Organization for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire (QLQ-C30). Initial compliance in the QoL study was 96% and the overall compliance 82%. QoL data were available for 245 patients (T 130 and 115 MF). Both treatment groups showed some improvement in emotional functioning during treatment, with a significant difference favouring the MF group at treatment cycles 5 and 6. In the T group, the scores on the other functional scales remained stable throughout the first six cycles. There were significant differences favouring the MF group on the social functioning scale at treatment cycle 6 and on the Global QoL scale at treatment cycles 5 and 6. On most symptom and single-item scales there were no statistically significant differences between the groups. However, at baseline, the T patients reported more appetite loss, at treatment cycles 2-4, the MF patients reported more nausea/vomiting, and at treatment cycle 6, the T patients reported more symptoms of fatigue, dyspnoea and insomnia. There were no statistically significant differences between the groups in the mean change scores of the functional and symptom scales. Interindividual variance was, however, larger in the T group. Differences in QoL between the two treatment groups were minor. Hence, given the expectancy of comparable QoL outcomes, the choice of treatment should be made on the basis of the expected clinical effect.

Structure-activity relationship of 2-[[(2-pyridyl)methyl]thio]-1H- benzimidazoles as anti Helicobacter pylori agents in vitro and evaluation of their in vivo efficacy.

A relationship between the structure of 21 2-[[(2-pyridyl)methyl]thio]-1H-benzimidazoles (6) and their anti Helicobacter pylori activity expressed as minimum bactericidal concentration (MBC) values is described. Observed MBCs ranged from 256 to 1 microg/mL. The structure-activity relationship (SAR) showed that larger and more lipophilic compounds, especially compounds with such substituents in the 4-position of the pyridyl moiety, generally had lower MBC values. Four new compounds that were predicted to be potent by the established SAR model were synthesized and tested. One such compound, i.e., 2-[[(4-[(cyclopropylmethyl)oxy]-3-methyl-2-pyridyl)methyl]thio]-1H-be nzimidazole (18), was tested for in vivo efficacy in a mouse Helicobacter felismodel (125 micromol/kg bid given orally for 4 days, n = 4). Unfortunately, antibacterial activity could not be clearly demonstrated in this model. Instead a potent acid secretion inhibition was observed. This finding was attributed to the methylthio compound being oxidized to the corresponding methyl sulfinyl derivative, i.e., a proton pump inhibitor, in vivo. Although the antibacterial activity had the potential of decreasing H. felis cell counts in vivo the proton pump inhibitory effect became dominant and actually promoted H. felis cell growth. Hence, we conclude that the antibacterial utility of the 2-[[(2-pyridyl)methyl]thio]-1H-benzimidazoles (6) as a compound class is compromised by their propensity to become proton pump inhibitors upon metabolic oxidation in vivo.

Factors affecting growth and antibiotic susceptibility of Helicobacter pylori: effect of pH and urea on the survival of a wild-type strain and a urease-deficient mutant.

This study investigated how pH and the presence of urea affect the survival and growth of Helicobacter pylori and whether these factors affect susceptibility to antibiotics in vitro. The viability of a wild-type strain and a urease-deficient mutant of H. pylori was studied after incubation for 1 h in buffers at different pH values at 37 degrees C under microaerophilic conditions. Viable counts were not affected at pH 5 and pH 7. In buffer at pH 3, there were no viable organisms, but urea (6.25 mM) protected the wild-type strain, which survived well. At pH 9, urea further reduced the viability of H. pylori and flurofamide almost abolished the effect of urea on the wild-type strain. Neither urea nor flurofamide affected the viability of the urease-deficient mutant under the same conditions. Growth was also pH dependent and was enhanced in shake-cultures. At pH 5, urea supported growth of the wild-type strain, but at pH 7 a toxic effect on the bacteria was observed. Growth of H. pylori at pH 5.9 was poor, and susceptibility to amoxycillin, erythromycin and clarithromycin was markedly less than at pH 7.2 and 7.9. The bactericidal activities of metronidazole and tetracycline were similar at the different pH values studied. At neutral pH the killing rates of amoxycillin and clarithromycin were growth rate dependent. Susceptibility to metronidazole was enhanced in stationary cultures. The interaction obtained between the proton pump inhibitor, omeprazole, and amoxycillin at pH 7 was of additive type. These results suggest that pH and growth conditions may be important in the antibacterial efficacy of different antibiotics in vivo and also provide a possible explanation for the potentiating effect of omeprazole with antibiotics in the treatment of H. pylori infections.

The dynamics of water in hydrated white bread investigated using quasielastic neutron scattering.

The dynamics of water in fresh and in rehydrated white bread is studied using quasielastic neutron scattering (QENS). A diffusion constant for water in fresh bread, without temperature gradients and with the use of a non-destructive technique, is presented here for the first time. The self-diffusion constant for fresh bread is estimated to be Ds = 3.8 × 10-10 m2 s-1 and the result agrees well with previous findings for similar systems. It is also suggested that water exhibits a faster dynamics than previously reported in the literature using equilibration of a hydration-level gradient monitored by vibrational spectroscopy. The temperature dependence of the dynamics of low hydration bread is also investigated for T = 280-350 K. The average relaxation time at constant momentum transfer (Q) shows an Arrhenius behavior in the temperature range investigated.

Cyclin A as a marker for prognosis and chemotherapy response in advanced breast cancer.

We wanted to study cyclin A as a marker for prognosis and chemotherapy response. A total of 283 women with metastatic breast cancer were initially enrolled in a randomised multicentre trial comparing docetaxel to sequential methotrexate-fluorouracil (MF) in advanced breast cancer after anthracycline failure. Paraffin-embedded blocks of the primary tumour were available for 96 patients (34%). The proportion of cells expressing cyclin A was determined by immunohistochemistry using a mouse monoclonal antibody to human cyclin A. Response evaluation was performed according to WHO recommendations. The median cyclin A positivity of tumour cells was 14.5% (range 1.2-45.0). Cyclin A correlated statistically significantly to all other tested proliferation markers (mitotic count, histological grade and Ki-67). A high cyclin A correlated significantly to a shorter time to first relapse, risk ratio (RR) 1.94 (95% CI 1.24-3.03) and survival from diagnosis, RR 2.49 (95% CI 1.45-4.29), cutoff point for high/low proliferation group 10.5%. Cyclin A did not correlate to chemotherapy response or survival after anthracycline, docetaxel or MF therapy. Of all tumour biological factors tested (mitotic count, histological grade and Ki-67), cyclin A seemed to have the strongest prognostic value. Cyclin A is a good marker for tumour proliferation and prognosis in breast cancer. In the present study, cyclin A did not predict chemotherapy response.

Factors affecting the biosynthesis of L-tryptophan by genetically modified strains of Escherichia coli.

Derivatives of Escherichia coli strain W3110 with increased tryptophan synthase (TS) activity were constructed. The biosynthesis of serine was shown to limit tryptophan production in minimal medium with indole as precursor. In the presence of serine and indole we obtained a correlation between the specific activity of TS and the specific productivity (qp) of tryptophan. Supplementation of the growth medium with glycine enhanced qp two-fold. In a strain with high serine hydroxymethyltransferase (SHMT) activity no such increase of tryptophan productivity was observed, although crude extracts from these cells were shown to produce tryptophan with indole, one-carbon units and glycine as precursors. Growth of the strain with high SHMT activity was inhibited in a medium with high glycine concentration. This inhibition could not be released by addition of isoleucine and valine. In a buffer system with permeabilized cells high in specific TS and SHMT activities we did not obtain any tryptophan production in presence of indole, glycine, one-carbon units and cofactors. On the other hand, in a buffer system with indole and serine as precursors we obtained high qp of tryptophan [13.3 g tryptophan (g dry wt cells)-1 h-1], which was correlated to the TS specific activity.

Role of the phi 11 phage genome in competence of Staphylococcus aureus.

Both phage ø11 and 83A, when present as prophage or when used as helper phage, induce competence for transfection and transformation to the same level in Staphylococcus aureus, strain 8325-4. Cells lysogenized with certain temperature-sensitive (ts) mutants of phage ø11 show competence at the nonpermissive temperature (41 C) without production of infectious phages. Phage ø11ts allele 31 can neither as a prophage nor as a helper phage develop competence under nonpermissive conditions. This mutant appears, therefore, to be mutated in the region of the phage genome controlling competence. The competence level for both transfection and transformation is increased by superinfecting strain 8325-4 (ø11) or 8325-4 (83A) at high multiplicities with phage ø11 with some of its mutants or with phage 83A. This superinfection enhancement appears to require protein synthesis but not deoxyribonucleic acid synthesis as judged from studies with inhibitors of macromolecular synthesis. Besides the phage particle, no extracellular or cell-bound factors so far detected can induce competence. The phage-induced product conferring competence is rapidly synthesized by strain 8325-4 (tsø11(31)) after shift to permissive conditions, but requires deoxyribonucleic acid and protein synthesis to be expressed. Recombination between the sus mutants of phage ø11 of Kretschmer and Egan and tsø11(31) indicate that competence is controlled by an early gene in the lytic cycle which may be expressed also in lysogenic cells. The phage product inducing competence appears to have a half-life of 10 to 15 min in the conditional lethal mutant at shift to nonpermissive temperature. Ultraviolet inactivation of phage ø11 infectivity occurs more rapidly than inactivation of competence induction. In fact, the number of transformants is increased at low doses of irradiation. Competence induction is, however, decreased at high does of ultraviolet irradiation.

Basis for the selective antibacterial activity in vitro of proton pump inhibitors against Helicobacter spp.

Proton pump inhibitors of the benzimidazole type exert a specific antibacterial activity against Helicobacter pylori in vitro. In the present study, the basis for this selectivity was investigated, and in particular, various factors affecting the in vitro antibacterial activity of sulfide analogs of benzimidazoles were studied. Upon preincubation of omeprazole for a period of up to 72 h in a buffer at pH 7, a product was formed that was bactericidal for H. pylori but had no effect on urease activity. Sulfide constitutes the main end product of degradation. The sulfide analog of omeprazole (H 168/22) exerted a bactericidal activity specifically against both resting (in buffer) and growing (in broth) Helicobacter spp., and time-kill in buffer at pH 5 was enhanced compared to that at pH 7. There was no or very low covalent binding of 3H-labeled H 168/22 to Helicobacter spp. or to other gram-negative and gram-positive bacteria. In the presence of fetal calf serum (FCS) under the same conditions, binding was only slightly lowered while the killing activity was markedly reduced, indicating a probably nonspecific interaction with proteins and/or protection of bacterial target(s) by FCS. Addition of H 168/22 (four times the minimum bactericidal concentration [MBC]) to exponentially growing H. pylori immediately stopped growth, and after an incubation period of 20 h viable counts were reduced by >7 log10. One-hour exposure of H. pylori to the drug followed by repeated washing retarded growth by about 2 h, indicating that the effect is reversible after short-term exposure. MICs and MBCs of various sulfide structures were lower than those obtained in broth after the addition of the corresponding sulfoxide. Thus, the MBC of the sulfide structure of omeprazole against 140 clinical isolates of H. pylori ranged from 8 to 32 microg/ml, compared to an MBC of omeprazole of 32 to 128 microg/ml. A similar potency was also recorded against other helicobacters. In conclusion, formation of sulfides of benzimidazoles in culture media is the reason for the selective antibacterial effect against H. pylori. The sulfides rapidly exerted a reversible antibacterial activity, which was specific against both resting and growing Helicobacter spp. without any covalent protein binding.

Biological characteristics of a type I restriction-modification system in Staphylococcus aureus.

Two restriction-modification systems, S1 and S2, are present in Staphylococcus aureus RN450 (S. Iordanescu and M. Surdeanu, J. Gen. Microbiol., 96:277-281, 1976). System S2 affects phage multiplication after both infection and transfection. Unmodified plasmid and chromosomal DNAs are also not expressed following transduction and transformation into a restrictive host. Restricted phages are, however, capable of conferring phage-mediated competence, although the state of competence does not affect the restriction-modification system. The restricting activity of system S2 is inactivated by heat treatment of the cells. An enzymatic activity that restricts unmodified phage DNA in the presence of ATP, Mg2+, and S-adenosylmethionine was recovered from cell-free extracts of a strain RN450 derivative.

Cloning of restriction fragments of DNA from staphylococcal bacteriophage phi 11.

EcoRI fragments of Staphylococcus aureus bacteriophage phi 11 DNA were cloned in vector plasmid pSA2100 in S. aureus. The clones were analyzed in marker rescue experiments with suppressor- and temperature-sensitive mutants of phi 11 to correlate the genetic and physical map. Several mutants could be identified on the physical map, and a clone containing fragment EcoRI-B of phi 11 DNA expressed immunity to phage infection. In addition, it was found that recombinant plasmids containing phi 11 DNA sequences can be transferred by high-frequency transduction after phage phi 11 infection of host cells.

Cytostar-T scintillating microplate assay for measurement of sodium-dependent bile acid uptake in transfected HEK-293 cells.

Real-time measurements of bile acid uptake into HEK-293 cell monolayers expressing the human sodium/bile acid cotransporters have been demonstrated using Cytostar-T microplates with an integral scintillating base. In these 96-well microplates, which permits culturing and observation of adherent cell monolayers, uptake of (14)C-labeled glycocholate and taurocholate into transfected HEK-293 cells was time-dependent, sodium-stimulated, and saturable. The sodium-activated uptake of 30 microM [(14)C]glycocholate (GC) via the ileal (IBAT) and liver (LBAT) transporters was 30-40 times higher than GC uptake in a sodium-free background. In addition, ouabain inhibition of the plasma membrane Na(+), K(+)-ATPase, causing the sodium gradient to collapse, resulted in total loss of glycocholate transport. Induction of gene expression by sodium butyrate showed that the amount of labeled bile acid accumulated in the cell monolayers at steady state was a function of the total amount of transporter expressed. Uptake of labeled bile acids was inhibited both by the specific IBAT inhibitor, 2164U90, and by various bile acids. No major difference was observed between IBAT and LBAT in their specificity for the bile acids tested while the dihydroxy bile acids had the highest affinity for both the transporters studied. The Cytostar-T proximity assay has been demonstrated to be an accurate and reproducible method for monitoring specific bile acid transport in transfected mammalian cells and the results are similar to those obtained by traditional methods. We conclude that the technique is an attractive approach to the cellular study of membrane transport of radiolabeled solutes in general and suggest a role in screening and characterization of novel transport inhibitors.