Clinical and microbiological characteristics of Klebsiella pneumoniae from community-acquired recurrent urinary tract infections.

Understanding the pathogenesis of recurrent urinary tract infection (RUTI) and whether it is attributable to reinfection with a new strain or relapse with the primary infecting strain is of considerable importance. Because previous studies regarding community-acquired Klebsiella pneumoniae RUTI are inconclusive, we undertook this study to evaluate the characteristics of the host and the bacterial agent K. pneumoniae in RUTI. A prospective study was designed, using consecutive patients diagnosed with community-acquired K. pneumoniae-related UTI from January 2007 to December 2009. Of the total 468 consecutive episodes, we found 7 patients with RUTI. All the patients with RUTI were elderly (median, 74 years), with diabetes (100 %, 7 out of 7). Clinical K. pneumoniae isolates derived from the same patients with RUTI revealed identical genomic fingerprints, indicating that K. pneumoniae UTI relapsed despite appropriate antibiotic therapy. The antimicrobial resistance, growth curve and biofilm formation of the recurrent isolates did not change. K. pneumoniae strains causing RUTI had more adhesion and invasiveness than the colonization strains (p < 0.01). When we compared the recurrent strains with the community-acquired UTI strains, the prevalence of diabetes mellitus was significant (100 % vs 53.7 %, p = 0.03) in the RUTI group. Our data suggest that K. pneumoniae strains might be able to persist within the urinary tract despite appropriate antibiotic treatment, and the greater adhesion and invasiveness in the recurrent strains may play an important role in recurrent infections.

Ischemic preconditioning reduces deep hypothermic circulatory arrest cardiopulmonary bypass induced lung injury.

PURPOSE: Ischemic preconditioning (IP) has been used to reduce ischemia-reperfusion injury in several models. It remains unknown whether IP is sufficient to prevent deep hypothermic circulatory arrest (DHCA) cardiopulmonary bypass (CPB) induced lung injury. MATERIALS AND METHODS: Twenty-four piglets were randomly divided into four groups: routine CPB (CPB), CPB + DHCA (DHCA), CPB + IP + DHCA (IP-1) and CPB + hypoxia-ischemia preconditioning + DHCA (IP-2). Lung static compliance (Cstat) and pulmonary vascular resistance (PVR) were measured as indicators of lung function at three points during CPB. TNF-α, IL-8 and IL-10 expressions were detected by radioimmunoassay. CD18 expression was determined by flow cytometer. Some lung tissues were excised to measure the wet/dry weight ratio (W/D) and some were fixed to observe pathological changes. RESULTS: Cstat significantly decreased whereas PVR increased in DHCA group. IP prevented DHCA-induced lung functional impairment, especially IP-2 treatment. More cytokines were produced after CPB in all groups, but with varying level. Left atrium/pulmonary artery ratio of CD18 expression on monocytes decreased only in DHCA group, whereas which on polymorphonuclear neutrophils decreased in DHCA group, IP-1 group at 1h post-CPB and IP-2 group. Although lung W/D was increased in IP-2 group compared with pre-CPB, but significantly lower than that in DHCA group. Histological findings showed less lung injuries in IP groups than DHCA group. CONCLUSIONS: DHCA aggravates lung inflammatory injury and IP may reverse this injury. Maintaining ventilation with pulmonary artery perfusion in the lung IP process during CPB seems to be more superior to single pulmonary artery perfusion.

Association of methionyl-tRNA synthetase with detergent-insoluble components of the rough endoplasmic reticulum.

Using fluorescent antibody staining, we have established the association of methionyl-tRNA synthetase with the endoplasmic reticulum in PtK2 cells. After Triton X-100 extraction, 70% of the recovered aminoacyl-tRNA synthetase activity was found in the detergent-insoluble fraction. This fraction of the enzyme remained localized with insoluble endoplasmic reticulum antigens and with ribosomes, which were stained with acridine orange. By both fluorescence microscopy and electron microscopy the organization of the detergent-insoluble residue was found to depend on the composition of the extracting solution. After extraction with a microtubule-stabilizing buffer containing EGTA, Triton X-100, and polyethylene glycol (Osburn, M., and K. Weber, 1977, Cell, 12:561-571) the ribosomes were aggregated in large clusters with remnants of membranes. After extraction with a buffer containing Triton X-100, sucrose, and CaCl2 (Fulton, A. B., K. M. Wang, and S. Penman, 1980, Cell, 20:849-857), the ribosomes were in small clusters and there were few morphologically recognizable membranes. In both cases the methionyl-tRNA synthetase and some endoplasmic reticulum antigens retained approximately their normal distribution in the cell. Double fluorochrome staining showed no morphological association of methionyl-tRNA synthetase with the microtubule, actin, or cytokeratin fiber systems of PtK2 cells. These observations demonstrate that detergent-insoluble cellular components, sometimes referred to as "cytoskeletal" preparations, contain significant amounts of nonfilamentous material including ribosomes, and membrane residue. Caution is required in speculating about intermolecular associations in such a complex cell fraction.

LncRNA HOTAIR inhibited osteogenic differentiation of BMSCs by regulating Wnt/ß-catenin pathway.

OBJECTIVE: This study aims to investigate whether HOX transcript antisense RNA (HOTAIR) can participate in the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by regulating the Wnt/ß-catenin pathway, thereby participating in the pathogenesis of osteoporosis. PATIENTS AND METHODS: We detected the expression level of HOTAIR in 60 osteoporosis patients and 60 normal controls by Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). Meanwhile, BMSCs derived from human or rats were subjected to determination of HOTAIR level. Subsequently, the effects of HOTAIR on osteogenic differentiation were evaluated by the activity of Alkaline Phosphatase (ALP), Alizarin Red S (ARS) staining, ALP staining and osteogenic-specific gene expression. The expression level of proteins related to the Wnt/ß-catenin was determined by Western blot, and ALP activity was detected by ALP activity determination kit and alizarin red staining after knockdown or overexpression of HOTAIR, as well as the treatment of DKK1 or the Wnt pathway antagonist. Finally, osteoporosis model in rats was established by ovariectomy (OVX). We examined protein levels of HOTAIR, ß-catenin, CyclinD, C-myc, and Runx2 in rat bone tissues. Bone morphology was observed in each group as well. RESULTS: The serum and BMSCs levels of HOTAIR in patients with osteoporosis were remarkably higher than that in normal people. Inhibition of HOTAIR induced increased ALP activity increased osteogenic marker genes and enhanced number of calcified nodules in BMSCs. However, the overexpression of HOTAIR exhibited the opposite effects. HOTAIR inhibited the expression level of Wnt/ß-catenin pathway-related protein. Also, Wnt pathway antagonist DKK1 partially reversed the regulatory effects of HOTAIR on Wnt/ß-catenin. DKK1 treatment markedly reduced the promotive effect of HOTAIR knockdown on ALP activity, ALP content and calcification ability of BMSCs. DKK1 administration in rats undergoing OVX showed worse bone morphology relative to controls. Protein levels of HOTAIR, ß-catenin, CyclinD, C-myc and Runx2 remarkably downregulated in OVX rats administrated with DKK1. CONCLUSIONS: HOTAIR inhibits osteoblast differentiation of rat BMSCs. The underlying mechanism of which may be related to the mediation of Wnt/ß-catenin pathway.

[Isolation of a novel fructose-1,6-bisphosphate aldolase gene from Codonopsis lanceolata and analysis of the response of this gene to abiotic stresses].

A cDNA clone containing a fructose-1,6-bisphosphate aldolase (ALD) gene, designated ClAldC, was isolated from a medicinal plant Codonopsis lanceolata. ClAldC is predicted to encode a precursor protein of 358 amino acid residues, and its sequence shares high degrees of homology with a number of other ALDs. The expression of ClAldC in different C. lanceolata organs was analyzed using reverse transcriptase (RT)-PCR. The results showed that ClAldC expressed high in stems of intact plant, while expressed at low level in leaves and roots. In addition, the expression of ClAldC under different abiotic stresses was analyzed at different time points. Three tested abiotic stimuli, anoxygenic stress, hydrogen peroxide and chilling, triggered a significant induction of ClAldC within 2-8 h post-treatment. However, there was no induction under other four stresses, NaCI, wounding, light and dark. The positive responses of ClAldC to the three abiotic stimuli suggested that C. lanceolata ClAldC may help to protect against environmental stresses such as anoxia, chilling and oxidative stress.

Generation of multiple forms of methionyl-tRNA synthetase from the multi-enzyme complex of mammalian aminoacyl-tRNA synthetases by endogenous proteolysis.

Methionyl-tRNA synthetase occurs free and as high-molecular-weight multi-enzyme complexes in rat liver. The free form is purified to near homogeneity by conventional column chromatography and affinity chromatography on tRNA-Sepharose. The native molecular weight of free methionyl-tRNA synthetase is 64 500, based on its sedimentation coefficient of 4.5 S and Stokes radius of 33 A. The free methionyl-tRNA synthetase apparently belongs to alpha-type subunit structure, since the subunit molecular weight is 68 000, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Methionyl-tRNA synthetase is dissociated from the high-molecular-weight synthetase complex by controlled trypsinization, according to Kellermann, O., Viel, C. and Waller, J.P. (Eur. J. Biochem. 88 (1978) 197-204). The dissociated, free methionyl-tRNA synthetase is subsequently purified to near homogeneity. The subunit structure of dissociated methionyl-tRNA synthetase is identical to that of endogenous free methionyl-tRNA synthetase. Anti-serum raised against Mr 104 000 protein in the synthetase complex, specifically inhibited methionyl-tRNA synthetase in both the free and the high-molecular-weight forms to the same extent. These results suggest that the occurrence of multiple forms of methionyl-tRNA synthetases in mammalian cells may, in part, be due to proteolytic cleavage.

Adenosine cyclic 3',5'-monophosphate-dependent protein kinase from human platelets.

A single cyclic AMP-dependent protein kinase (EC 2.7.1.37) has been isolated from human platelets by using DEAE-cellulose ion-exchange chromatography and Sephadex G-150 gel filtration. The molecular weight of the protein kinase was estimated to be 86 490. In the presence of cyclic AMP, the protein kinase could be dissociated into a catalytic subunit of molecular weight 50 000, and either one regulatory subunit of molecular weight 110 000 or two regulatory subunits of molecular weights 110 000 and 38 100, depending on the pH used. Recombination of either of the regulatory subunits with the catalytic subunit restored cyclic AMP-dependency in the catalytic subunit. The apparent Km for ATP in the presence of 10 muM Mg2+ was 4 muM (plus cyclic AMP) and 4.3 muM (minus cyclic AMP). The concentration of cyclic AMP needed for half-maximal stimulation of the protein kinase was 0.172 muM and apparent dissociation constants of 3.7 nM (absence of MgATP) and 0.18 muM (presence of MgATP) were exhibited by the "protein kinase-cyclic AMP complex". The enzyme required Mg2+ for maximum activity and showed a pH optimum of 6.2 with histone as substrate. In addition to four major endogenous platelet protein acceptors of apparent molecular weights 45 000, 28000, 18 500, and 11 100, the platelet protein kinase also phosphorylated the exogenous acceptor proteins thrombin, collagen and histone, all capable of inducing platelet aggregation. Prothrombin, a nonaggregating agent, was not phosphorylated.

Interactions of aminoacyl-tRNA synthetases in high-molecular-weight multienzyme complexes from rat liver.

The functional interaction of Arg-, Ile-, Leu-, Lys- and Met-tRNA synthetases occurring within the same rat liver multienzyme complex are investigated by examining the enzymes catalytic activities and inactivation kinetics. The Michaelis constants for amino acids, ATP and tRNAs of the dissociated aminoacyl-tRNA synthetases are not significantly different from those of the high-Mr multienzyme complex, except in a few cases where the Km values of the dissociated enzymes are higher than those of the high-Mr form. The maximal aminoacylation velocities of the individual aminoacyl-tRNA synthetases are not affected by the presence of simultaneous aminoacylation by another synthetase occurring within the same multienzyme complex. Site-specific oxidative modification by ascorbate and nonspecific thermal inactivation of synthetases in the purified rat liver 18 S synthetase complex are examined. Lys- and Arg-tRNA synthetases show remarkably parallel time-courses in both inactivation processes. Leu- and Met-tRNA synthetases also show parallel kinetics in thermal inactivation and possibly oxidative inactivation. Ile-tRNA synthetase shows little inactivation in either process. The oxidative inactivation of Lys- and Arg-tRNA synthetases can be reversed by addition of dithiothreitol. These results suggest that synthetases within the same high-Mr complex catalyze aminoacylation reactions independently; however, the stabilities of some of the synthetases in the multienzyme complex are coupled. In particular, the stability of Arg-tRNA synthetase depends appreciably on its association with fully active Lys-tRNA synthetase.

Cloning, sequencing and expression of a cDNA encoding mammalian valyl-tRNA synthetase.

A fragment of the cDNA encoding a rat valyl-tRNA synthetase (TrsVal)-like protein was cloned from a rat cDNA library in lambda gt11 using an oligodeoxyribonucleotide (oligo) probe. Three independent plaque clones containing the human TrsVal cDNA were then isolated from a lambda gt10 human erythroleukemia cDNA library using the rat cDNA fragment as the hybridization probe. Sequence analyses of the cDNA fragments provided a 3.2-kb sequence with an open reading frame that contained the 'HIGH' synthetase signature sequence and the tRNA 3'-end-binding motif, KMSKS, and putative Val-binding motif, EWCISRQ. The sequence was extended to the 3' end of the cDNA by the polymerase chain reaction using an internal primer and an oligo(dT) adapter. The deduced 1051-amino-acid sequence shares 65% identity with yeast TrsVal, and contains a highly basic N-terminal region, a newly evolved protease-sensitive region in sequence close to the C terminus, and several sites for protein kinase C phosphorylation. A 3-kb cDNA fragment was sub-cloned into plasmid pSVL and expressed in COS-7 cells; up to a sevenfold increase in TrsVal activity was obtained. These results confirm the cloning and sequencing of a human TrsVal-encoding cDNA.

Proteolytic signal sequences (PEST) in the mammalian aminoacyl-tRNA synthetase complex.

Eight aminoacyl-tRNA synthetases together with three unidentified proteins are associated as a multi-enzyme complex in mammalian cells. Partial peptide sequences for lysyl- and aspartyl-tRNA synthetases are determined and no highly hydrophobic peptides are found. The partial amino acid sequences for two of the unidentified proteins in the complex are shown to have substantial homology and each has a number of unique sequences. The results suggest that the two unidentified proteins are fragments of synthetases. The partial sequences revealed the presence of PEST sequences in at least three proteins. Inasmuch as PEST sequences are signals for intracellular degradation, the mammalian synthetase complex may have evolved to protect these synthetases against intracellular proteolysis.

Some analogues of luteinizing hormone-releasing hormone with substituents in position 10.

As part of our studies on the design of agonists of the luteinizing hormone-releasing hormone (LH-RH), we have synthesized the [des-Gly-NH2(10)]-LH-RH N-methylhydrazide (1), the corresponding thiosemicarbazide (2), and the N-formyl- (3) N-acetyl- (4) and N-(trifluoroacetyl)hydrazide (5). Analogue 1 may be regarded as isosteric with [des-Gly-NH2(10)]-LH-RH N-alkylamides which are, in general, potent agonists. Analogues 2-5 may be regarded as isosteric with [aza-Gly-NH2(10)]-OH-RH, which is equipotent with the hormone. The required protected intermediates were prepared by solid-phase synthesis, and the free peptides were prepared from them by deprotection with HF, followed by purification on Sephadex G-25. Bioassay of these analogues with rat hemipituitaries in vitro showed the following values as percentages of the hormonal values for the release of LH and FSH respectively: N-methylhydrazide (1), 17 and 11%; semithiocarbazide (2), 6.5 and 4.6%; N-formylhydrazide (3), 15.3 and 10%; N-acetylhydrazide (4), 1.2 and 0.6%; N-(trifluoroacetyl)hydrazide (5), 1.0 and 0.9%. Thus, these types of isosteric substitutions are inimical to the preservation of the high biological activity of LH-RH.

An approach to the elucidation of metabolic breakdown products of the luteinizing hormone-releasing hormone.

Metabolic breakdown of the luteinizing hormone-releasing hormone (LH-RH) could lead to the following fragments containing pyroglutamic acid: pyroglutamic acid (1), pGlu-His (2), pGLu-His-Trp (3), pGlu-His-Trp-Ser (4), etc., and finally pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly (10). We have synthesized fragments 2-10 and successfully separated all ten metabolites and LH-RH by high-performance liquid chromatography (HPLC) with a muBondapak C18 column. In a test of the viability of the method, cochromatography of fragments 1-10 and LH-RH with the products of chymotryptic digestion of tritiated LH-RH showed radioactive peaks corresponding to the expected products, fragments 3 and 5. Analysis of the products of incubation of a rat kidney homogenate supernatant with LH-RH showed fragments 1-4 and LH-RH. The finding of breakdown at position 4 uncovers a new site of LH-RH breakdown and points the way to the design of potential LH-RH antagonists and agonists where the 4 position would be substituted with unnatural amino acids to prevent breakdown.

Selective cyclooxygenase-2 inhibitors: heteroaryl modified 1,2-diarylimidazoles are potent, orally active antiinflammatory agents.

A series of heteroaryl modified 1,2-diarylimidazoles has been synthesized and found to be potent and highly selective (1000-9000-fold) inhibitors of the human COX-2. 3-Pyridyl derived COX-2 selective inhibitor (25) exhibited excellent activity in acute (carrageenan induced paw edema, ED(50) = 5.4 mg/kg) and chronic (adjuvant induced arthritis, ED(50) = 0.25 mg/kg) models of inflammation. The relatively long half-life of 25 in rat and dog prompted investigation of the pyridyl and other heteroaromatic systems containing potential metabolic functionalities. A number of substituted pyridyl and thiazole containing compounds (e.g., 44, 46, 54, 76, and 78) demonstrated excellent oral activity in every efficacy model evaluated. Several orally active diarylimidazoles exhibited desirable pharmacokinetics profiles and showed no GI toxicity in the rat up to 100 mg/kg in both acute and chronic models. The paper describes facile and practical syntheses of the targeted diarylimidazoles. The structure-activity relationships and antiinflammatory properties of a series of diarylimidazoles are discussed.

Characterization of metabolites of Celecoxib in rabbits by liquid chromatography/tandem mass spectrometry.

The metabolism of the anti-inflammatory drug Celecoxib in rabbits was characterized using liquid chromatography (LC)/tandem mass spectrometry (MS/MS) with precursor ion and constant neutral loss scans followed by product ion scans. After separation by on-line liquid chromatography, the crude urine samples and plasma and fecal extracts were analyzed with turbo-ionspray ionization in negative ion mode using a precursor ion scan of m/z 69 (CF(3)) and a neutral loss scan of 176 (dehydroglucuronic acid). The subsequent product ion scans of the [M - H] ions of these metabolites yielded the identification of three phase I and four phase II metabolites. The phase I metabolites had hydroxylations at the methyl group or on the phenyl ring of Celecoxib, and the subsequent oxidation product of the hydroxymethyl metabolite formed the carboxylic acid metabolite. The phase II metabolites included four positional isomers of acyl glucuronide conjugates of the carboxylic acid metabolite. These positional isomers were caused by the alkaline pH of the rabbit urine and were not found in rabbit plasma. The chemical structures of the metabolites were characterized by interpretation of their product ion spectra and comparison of their LC retention times and the product ion spectra with those of the authentic synthesized standards.

5-HT4 receptor activation induces relaxation and associated cAMP generation in rat esophagus.

Changes in mechanical events and intracellular levels of cAMP induced by the activation of the 5-HT4 receptor were investigated in the rat esophagus tunica muscularis mucosae preparation. Serotonin (5-HT) and 5 methoxytryptamine (5-MOT; 5-HT4 agonist) caused concentration-related relaxation responses, while 5-carboxamidotryptamine (5-CT; 5-HT1 agonist), 1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane (DOI; 5-HT2 agonist) and 2-methyl-serotonin (2-methyl-5-HT; 5-HT3 agonist) were less active. The prokinetic agents, cisapride and renzapride also induced concentration-dependent relaxation of rat esophagus which was intermediate to 5-HT and 5-MOT in potency. The relaxation was not due to activity at receptors other than the 5-HT4 since methysergide (5-HT1 and 5-HT2 antagonist) and granisetron (5-HT3 antagonist) did not block the relaxant response to 5-HT while ICS 205930 (5-HT4 antagonist) antagonized this response (pA2 = 6.45). Serotonin also caused concentration-related increases in tunica muscularis mucosae cAMP with the rank order of efficacy of 5-HT agonists in raising tissue cAMP levels reflecting their relaxant activities (5HT greater than or equal to 5-MOT greater than 5-CT greater than DOI = 2-methyl-5-HT = control). Enhancement of cAMP concentrations was also observed following renzapride treatment. This cAMP relaxation response was specific for 5-HT4 receptor activation as demonstrated by the lack of ICS 205930 inhibition of rat esophagus relaxation caused by isoproterenol, 16,16-dimethyl-prostaglandin E2 and forskolin.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of transferrin receptor and ferritin H-chain mRNA are associated with clinical and histopathological prognostic indicators in breast cancer.

It is well known that iron plays an essential role in many biochemical reactions and that rapidly growing cells require more iron for their growth and metabolism than resting cells. Transferrin and its receptor are required for entry of iron into the cell. In contrast, ferritin is a cellular storage protein whose main function is to sequester excess ferric iron and thus prevent high concentrations of soluble ferric iron from becoming toxic to the cell. However, the clinical significance of both transferrin receptor and ferritin mRNA levels have not previously been described in tumors from breast cancer patients. In this study, tumor tissue mRNA levels of transferrin receptor and ferritin were quantitated on forty-two breast cancer patients. A highly sensitive non-radioisotopic cDNA polymerase chain reaction assay was used to quantitate expression of mRNA. The expression of glyceraldehyde-3-phosphate dehydrogenase served as the control. In the tumor tissue from the 42 breast cancer patients the transferrin receptor mRNA levels were significantly correlated to the ferritin H-chain mRNA levels (Spearman correlation r = 0.5433, p = 0.0002; Pearson correlation r = 0.6276, p < 0.0001). The level of amplified transferrin receptor complementary DNA was related to differentiation (ANOVA, p = 0.042) with poorly differentiated tumors having high levels of transferrin receptor mRNA. Further, the levels of amplified gene for ferritin heavy chain complementary DNA was directly related to axillary lymph nodes status (Student's t-test, p = 0.044), presence of metastatic disease (Student's t-test, p = 0.046) and clinical stage (stage I + stage II versus stage III + stage IV; Student's t-test, p = 0.0181). These results demonstrate that non-radioisotopic RT-PCR is a very sensitive method for determining mRNA levels in tumor tissue. Additionally, the quantitation of expression of transferrin receptor and ferritin heavy chain mRNA may be useful for assessing prognosis and guiding therapeutic decisions in breast cancer patients.

Doxorubicin-gallium-transferrin conjugate overcomes multidrug resistance: evidence for drug accumulation in the nucleus of drug resistant MCF-7/ADR cells.

Development of multidrug resistance (MDR) in cancer cells decreases net doxorubicin (ADR) uptake as a result of increased efflux, increased intracellular sequestration, and decreased membrane permeability. In this study, we investigated whether conjugation of ADR to transferrin (Tf) could overcome MDR in breast cancer cells. The multidrug resistant MCF-7/ADR breast cancer cell line was over 1000-fold more resistant to ADR, than its parental MCF-7 cell line, as determined by 3[H]-thymidine assay. The MCF-7/ADR cell line also expressed both MDR1 and MRP genes, as detected by RT-PCR. The ADR was coupled using a glutaraldehyde technique to human transferrin saturated with either ferric chloride (Fe-Tf) or gallium nitrate (Ga-Tf). These conjugates were tested for cytotoxicity on both MCF-7 and MCF-7/ADR cells after 6 days of incubation. The doxorubicin-gallium-transferrin conjugate (ADR-Ga-Tf) exhibited approximately the same inhibitory effect as ADR on MCF-7 cells with IC50s of 2.34 x 10(-3) microM and 1.42 x 10(-3) microM, respectively. However in MCF-7/ADR cells ADR-Ga-Tf reversed resistance to free ADR and decreased 100-fold the IC50 from 8.98 microM with free ADR to 9.52 x 10(-2) microM. ADR-Fe-Tf was 10-fold more inhibitory to MCF-7/ADR cells than free ADR. Compared to Ga-Tf, ADR-Ga-Tf was 500- and 3000-fold more inhibitory to MCF-7 and MCF-7/ADR cells, respectively. These results demonstrated that ADR-Ga-Tf reverses resistance to free ADR and Ga-Tf in MCF-7/ADR cells. The distribution of ADR in both cell lines was examined by fluorescence microscopy. It was noted that ADR mainly accumulated in the cytoplasm around the nucleus in MCF-7/ADR cells, but in both the cytoplasm and nucleus of MCF-7 cells. However the conjugate of ADR-Ga-Tf allowed ADR to accumulate in the cytoplasm and nucleus of both the MCF-7/ADR and MCF-7 cells. Further investigation of MDR1 and MRP genes expression by RT-PCR demonstrated that Ga-Tf decreased expression of the MRP more than the MDR1 gene. Therefore the reversal of resistance to ADR by the ADR-Ga-Tf conjugate is mediated by the transferrin receptor transmembrane transport mechanism, redistribution of ADR into the nucleus of ADR resistant MCF-7/ADR cells and inhibition of MRP gene expression.

Inhibition of growth of human breast carcinoma cells by an antisense oligonucleotide targeted to the transferrin receptor gene.

Transferrin receptor expression is controlled by the amount of iron required by the cell to maintain its metabolism and therefore tumor cells in a highly proliferative state have a high density of transferrin receptors. In this study, phosphorothioated antisense TfR oligonucleotides (TfR-ODna) targeted to the sequences of TfR mRNA including the AUG initiation codon and the control sense chain (TfR-ODns) were synthesized. The rate of cellular DNA synthesis was determined by [3H]-thymidine incorporation. Administering TfR-ODna to three morphologically distinct breast cancer cell lines (MCF-7, T47D, and MDA-MB-231) and a normal breast cell line (MCF-12A) caused specific inhibition of tumor cell growth. The IC50 (50% inhibition of DNA synthesis) of the TfR-ODna for the MCF-7, T47D and MDA-MB-231 cells were 0.5, 0.5, and 1.0 microM, respectively, whereas the MCF-12A normal breast cells were about 30 times (IC50 of 30 microM) less sensitive to TfR-ODna than the breast cancer cells. The cytotoxicity of the antisense TfR-ODna was 10 to 60 times greater than that of the sense chain (TfR-ODns). TfR mRNA and protein synthesis were demonstrated by RT-PCR and immunohistochemical staining, respectively. Approximately 50% inhibition of the expression of TfR mRNA was observed when breast cancer cells were treated with 1 microM antisense TfR ODNa for 72 hrs but 1 microM antisense only caused 14% inhibition in normal breast cells. The decreased cytotoxicity and inhibition of TfR gene expression when the tumor cells were treated with the same concentration (1 microM) of TfR-ODns demonstrated the specificity of the TfR-ODna for blocking the target TfR gene. The combined cytotoxicities to human breast tumor MCF-7 cells of the antisense TfR-ODna and the iron chelator deferoxamine (DFO) or the ribonucleotide reductase inhibitor hydroxyurea were observed in this study. IC50s (50% inhibition of DNA synthesis) for DFO and hydroxyurea individually were 0.3 microM and 250 microM, respectively. The CalcuSyn program was used to determine the combined effects among the agents and synergism (Combined Indexes (CI) < 1) were found with the following two combinations: TfR-ODna (0.007 microM to 0.15 microM) with DFO (0.15 microM to 5 microM) and TfR-ODna (0.007 microM to 0.15 microM) with hydroxyurea (50 microM to 800 microM). However, inhibition by TfR-ODns was not synergistic with either DFO or hydroxyurea. The synergistic effects on inhibition of DNA synthesis between TfR-ODna and DFO or hydroxyurea suggest that inhibition of breast cancer cell growth by TfR-ODna is produced by depletion of iron pools that are required for DNA synthesis in tumor cells. The fact that TfR-ODna specifically decreases cell viability and proliferation, and reduces TfR mRNA and protein expression in human breast carcinoma cells without affecting normal breast cells, suggests that the antisense oligonucleotide to the transferrin receptor may be a novel therapeutic approach in breast cancer.

Vaccination with a mixed vaccine of autogenous and allogeneic breast cancer cells and tumor associated antigens CA15-3, CEA and CA125--results in immune and clinical responses in breast cancer patients.

In breast cancer there is often overexpression of the breast cancer antigen CA15-3, the carcinoembryonic antigen (CEA) and the ovarian cancer antigen CA125, which makes them potential target antigens for immunotherapy. In this study, we used a multi-antigen vaccine, which included the following antigens: autologous breast cancer cells (AUTOC), allogeneic breast cancer MCF-7 cells (ALLOC), and the tumor associated antigens CA15-3, CEA and CA125, plus low doses of granulocyte/macrophage-colony-stimulating factor (GM-CSF) and interleukin 2 (IL-2). Forty-two breast cancer patients received weekly subcutaneous vaccination at the 1st, 2nd, 3rd, 7th, 11th and 15th weeks. Their lymphocyte proliferative responses to AUTOC, ALLOC, CA15-3, CEA and CA125 were tested in lymphocyte blastogenesis assays (LBA) before and after vaccination. The disease stage and serum CA15-3, CEA and CA125 concentrations were also determined pre- and post-vaccination. We found that the vaccine was safe, and the only major side effects were swelling at the site of injection, muscle pain, and weakness or fatigue. The vaccine induced a significant increase in post-vaccination lymphocyte proliferative responses to AUTOC, CA15-3, CEA and CA125 but not ALLOC, compared to pre-vaccination (p < 0.05, p < 0.01, p < 0.05, p < 0.01 and p > 0.05, respectively, a paired t Test). Computed tomography (CT), ultrasound or bone scan showed evidence of disease improvement in 2 (12%) patients after vaccination. Hepatic metastases were reduced in size and number and some actually disappeared one patient. Metastatic disease in the L5 vertebra and the skull decreased in size and some osteolytic sites completely healed in a second patient. In addition, 7 patients (44%) had stable disease and 7 patients (44%) had disease progression. We did not find vaccination significantly reduced serum tumor markers CA15-3, CEA and CA125 of these breast cancer patients. These results suggest that the vaccine mixture of autologous and allogeneic breast cancer cells and tumor associated antigens plus GM-CSF and IL-2 can be administered safely to breast cancer patients and there is evidence for improved immunity and clinical efficacy.