PubMed Health⌕ Search

Biomedical subjects

J Koropatnick

Publications and source records attributed to J Koropatnick.

At least 19 recordsLinked to original sources
PubMed1999

Differential expression of PSP94 in rat prostate lobes as demonstrated by an antibody against recombinant GST-PSP94.

Prostate secretory protein (PSP94, 94 amino acids) is one of the most abundant proteins secreted from the prostate. Its biological role is unknown and still controversial, although it is assumed to have the potential to be a biomarker and a suppressor of prostate cancer. In order to establish an animal model to further elucidate its biological role, we expressed the mature form of rat PSP94 in Escherichia coli, using a glutathione S-transferase (GST) fusion expression vector; we generated a polyclonal rabbit antibody against the recombinant protein. The antibody specifically recognized recombinant rat PSP94 and cross-reacted only very weakly with its human homologue. Using the characterized anti-rat PSP94 antibody, we found that PSP94 was located primarily in rat prostate. Furthermore, PSP94 is present at different levels in different lobes of rat prostate, with significant levels detectable only in the lateral lobe (LP). In addition, the most abundant PSP94 expression was found in the prostate lobe secretions, and PSP94 levels in LP secretions were at least seven times higher than in secretions from the dorsal prostate (DP). The rat ventral prostate (VP) and other regions of the male accessory glands were found to be almost completely devoid of PSP94. Since most rat prostate dysplasia induced by steroid hormone treatment occurs only in dorsolateral prostate, prostate tissue-specific expression and the expression of PSP94 in dorsolateral, but not other, lobes of the prostate suggest a potential role in prostate targeting and prostate cancer development.

Animals↗
PubMed1999

Antisense down-regulation of thymidylate synthase to suppress growth and enhance cytotoxicity of 5-FUdR, 5-FU and Tomudex in HeLa cells.

1. Thymidylate synthase (TS), the key enzyme in de novo synthesis of thymidine, is an important target for antitumour chemotherapy. It was hypothesized that antisense oligonucleotide down-regulation of TS mRNA would decrease TS levels and enhance the cytotoxicity of inhibitors of TS, including the pyrimidine analogues 5-fluorouracil (5-FU) and 5-fluorodeoxyuridine (5-FUdR), and the folate analogue Tomudex (ICI D1694; N-(5-[N-(3, 4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino ]-2-theon yl-L-glutamic acid). 2. 2'-Methoxyethoxylated, phosphorothioated 20-mer oligodeoxynucleotides (ODNs), complementary to various sequences in TS mRNA, were synthesized, along with control oligomers consisting of the same, respective bases in randomized order, against which all the biological effects were compared. Following a 6-h transfection of HeLa cells using polycationic liposome at 3 microg ml(-1), ODN 83 (50 nM), complementary to a region in the 3'-untranslated region of the TS mRNA, decreased TS mRNA levels by approximately 70% within 24 h. ODN 83 also decreased TS enzyme activity, as measured by binding of TS to radiolabelled 5-fluorodeoxyuridine monophosphate. In addition to inhibiting proliferation by up to approximately 40%, ODN 83 enhanced the cytotoxicity of Tomudex or 5-FU, added 1 day following transfection, by 50 - 60%. ODN 83 also enhanced sensitivity to 5-FUdR by 70%, but did not affect the toxicity of cisplatin, chlorambucil, melphalan, doxorubicin, ionizing radiation, paclitaxel, or irinotecan. 3. These data indicate that antisense ODN down-regulation of TS can inhibit human tumour cell proliferation and enhance the efficacy of TS-targeted drugs.

Antimetabolites, Antineoplastic↗
PubMed1998

Antisense nucleic acids targeted to the thymidylate synthase (TS) mRNA translation start site stimulate TS gene transcription.

Thymidylate synthase (TS) is a key enzyme in the synthesis of DNA and a target for cancer chemotherapeutic agents. Antisense TS nucleic acids may be useful in enhancing anticancer drug effectiveness. MCF-7 and HeLa cells were transfected with vectors expressing antisense TS RNA or with antisense oligodeoxynucleotides (AS-ODNs) to different TS mRNA regions. Antisense RNAs were targeted to 30 bases of the TS mRNA including part of the stem loop at the translation start site and to 30 bases spanning the exon1/exon2 boundary. AS-ODNs were targeted to the translation start site and the translation stop site. Antisense nucleic acids complementary to the translation start site (and not the exon1/exon2 boundary or translation stop site) significantly enhanced constitutive TS gene transcription. Therefore, TS mRNA sequences appear to be involved in a novel pathway controlling TS gene transcription. Induced transcription could hinder antisense-based attempts to inhibit TS and must be considered when designing such strategies.

Blotting, Northern↗
PubMed1998

Metallothionein and apoptosis in the toxic milk mutant mouse.

Toxic milk mutant (tx) mice accumulate excess copper (Cu) in liver with age and develop symptoms similar to those seen in human Wilson disease. Because metallothionein (MT) is the major Cu-binding protein in tx mouse liver and Cu-MT can enhance lipid peroxidation initiated by an organic hydroperoxide, the potential genotoxicity of Cu-MT in tx mice was assessed in male tx mice (11 to 12 months old) and in age- and sex-matched control wild-type (DL) mice. Toxic milk mutant mice, but not control DL mice, developed regenerative liver nodules (tx-N) with normal histologic appearance. Residual, non-nodular tx mouse liver (tx-R) was microscopically abnormal with large, atypical hepatocytes. The levels of Cu, zinc (Zn), and MT, and the numbers of apoptotic cells (APC) in tx-N, tx-R, and DL livers were measured by atomic absorption spectrophotometry, 109cadmium-heme assay, and the TUNEL method, respectively. Significantly higher levels of MT, Cu, and Zn, as well as increased numbers of APC were found in both tx-N and tx-R compared with DL mouse livers. Intense nuclear and cytoplasmic immunohistochemical staining for MT was observed in both normal and atypical hepatocytes of the tx mouse, whereas only cytoplasmic staining for MT was detected in DL mouse liver tissue. Accumulated Cu could be detected in tx-R and tx-N liver by rhodanine staining but was not detected in other tx mouse organs, or in mouse liver or other organs of DL. The number of APC and level of MT were significantly higher in tx-R liver compared with both tx-N and DL liver. These results suggest that: (a) aged tx mouse accumulate excess Cu in liver accompanied by striking morphologic changes, and (b) although MT binds to Cu in tx mouse liver, the presence of high Cu-MT and Cu in the nucleus can be genotoxic and may lead to enhanced apoptosis.

Animals↗
PubMed1997

Effect of non-toxic mercury, zinc or cadmium pretreatment on the capacity of human monocytes to undergo lipopolysaccharide-induced activation.

1. Metal salts can inhibit cell activity through direct toxicity to critical cellular molecules and structures. On the other hand, they can also change cell behaviour by inducing specific genes (including genes encoding members of the metallothionein [MT] gene family). Therefore, transition metals may affect cell functions either by acting as a toxin, or by transmitting or influencing signals controlling gene expression. 2. To explore the latter possibility, we measured the ability of low, non-toxic metal pretreatment to alter immune cell behaviour. We previously found that pretreatment of human monocytes with zinc induces metallothionein gene expression and alters their capacity to undergo a bacterial lipopolysaccharide-induced respiratory burst. We showed here that cadmium and mercury salts, at concentrations that exert no discernible toxicity, inhibit activation of human monocytic leukemia (THP-1) cells. CdCl2 1 microM, ZnCl2 20-40 microM or HgCl2 2 microM pretreatment for 20 h induced MT-2 mRNA and total MT protein accumulation and had no effect on proliferation potential or metabolic activity, but significantly inhibited the ability of subsequent lipopolysaccharide treatment to induce the oxidative burst, increased adhesion to plastic, and MT-2 and interleukin-1 beta (IL-1 beta) mRNA accumulation. 3. The phenomenon of metal-induced suppression of monocyte activation, at metal concentrations that have no effect on cell viability, has important implications for assessment of acceptable levels of human exposure to cadmium, zinc and mercury.

Cadmium↗
PubMed1995

Metallothionein protects DNA from copper-induced but not iron-induced cleavage in vitro.

Iron and copper ions mediate generation of reactive oxygen radicals from O2 and H2O2 by the Fenton reaction: these radicals are capable of damaging DNA. We studied (a) the ability of these metals to induce double-strand breaks in DNA in vitro in the presence of H2O2 and ascorbic acid as donors of reactive oxygen, and (b) the ability of the metal-binding protein metallothionein (MT) to protect DNA from damage. Strand cleavage was measured by loss of fluorescence after binding to ethidium bromide and by increased mobility of DNA in agarose. The results show that Cu(II), Fe(II) and Fe(III) all can induce damage to calf thymus DNA under our experimental conditions. Cu(II)-induced DNA damage was dose-dependent and the degree of damage was proportional to the concentration of H2O2. On the other hand, DNA fragmentation was significant only in the presence of high concentrations of Fe(II) or Fe(III). Addition of Zn-MT to the reaction mixture prior to addition of Cu(II) inhibited fragmentation of DNA in a dose-dependent manner but had little effect on iron induced damage. Other proteins (histone or albumin) were not effective in protecting DNA from Cu-induced damage, as compared to Zn-MT. The formation of Cu(I) from Cu(II) in the presence of hydrogen peroxide and ascorbate was also inhibited by addition of Zn-MT. Thus, MT may protect DNA from damage by free radicals by sequestering copper and preventing its participation in redox reactions.

Animals↗
PubMed1995

Sensitivity to cadmium-induced genotoxicity in rat testicular cells is associated with minimal expression of the metallothionein gene.

Cadmium is a carcinogenic metal. Although the mechanism of tumor induction is unknown, DNA/metal interactions may be involved. Metallothionein can protect against cadmium toxicity in our previous work it was shown to reduce cadmium genotoxicity in cultured cells. To extend these results, the genotoxicity of cadmium was studied in R2C cells, a rat testicular Leydig cell line. The R2C cells were very sensitive to cadmium-induced single-strand DNA damage (SSD), as measured by alkaline elution. SSD occurred in R2C cells after treatment with 25 and 50 microM CdCl2 for 2 hr. Prior work showed other cells required much higher levels of cadmium (approximately 500 microM) to induce genotoxicity. The genotoxic levels of cadmium (25-50 microM) were not cytotoxic in R2C cells as assessed by a metabolic activity (MTT) assay. Pretreatment of R2C cells with a low cadmium dose (2 microM, 24 hr) had no effect on cadmium-induced SSD, in contrast to prior work in other cells where such pretreatments reduced SSD through metallothionein gene activation. In fact, cadmium or zinc treatments resulted in little or no increase in metallothionein gene expression in R2C cells as determined by Northern blot analysis for metallothionein mRNA using cDNA or oligonucleotide probes and radioimmunoassay for metallothionein protein production. Basal metallothionein mRNA was essentially nondetectable. Induction of a cadmium-binding protein in R2C cells did occur, as determined by Cd-heme assay, but did not induce tolerance to SSD. In vivo, the Leydig cell is a target for cadmium carcinogenicity and its cadmium-binding protein is thought not to be a true metallothionein. These results indicate that R2C cells are sensitive to cadmium-induced genotoxicity and that this sensitivity is associated with minimal expression of the metallothionein gene.

Animals↗
PubMed1995

Enhanced transcription of metallothionein genes in rat kidney: effect of uninephrectomy and compensatory renal growth.

Metallothioneins (MTs) have been implicated in the intracellular regulation of essential metals in eukaryotic cells, and increased expression of MT genes has been demonstrated during the growth and proliferation of cells. To explore the expression of MT in somatic cells undergoing growth (hypertrophy) in the kidney in situ, we measured the rates of transcription of the genes for MT-1 and MT-2, measured the levels of mRNA for MT-1 and MT-2, and measured the concentration of MT-1 and MT-2 protein in samples of renal (and hepatic) tissue from uninephrectomized (NPX) and sham-operated (SO) rats 15 days after surgery. The rates of transcription of the genes for MT-1 and MT-2 were found to be enhanced significantly in the remnant renal mass, particularly in the cortex and outer stripe of the outer medulla, and in the liver, after uninephrectomy and after 15 days allowing for compensatory renal growth. Increased accumulation of mRNA for MT-1 and MT-2 also occurred in the cortex and outer stripe of the outer medulla of the remnant kidney and in the liver in the NPX rats. Increased concentration of MT-1 and MT-2 protein (measured by radioimmunoassay), at the level of the whole kidney, renal cortex, and liver, was another feature detected in rats after uninephrectomy and 15 days of compensatory renal growth. These findings indicate that compensatory renal growth in response to uninephrectomy is associated with the induction of the expression of MT genes in the renal cortex and outer stripe of the outer medulla, as well as in the liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptation, Physiological↗
PubMed1995

Metallothionein expression and resistance to cisplatin in a human germ cell tumor cell line.

Expression of intracellular metallothionein (MT) has been linked to cis-diamminedichloroplatinum (cDDP) resistance in human germ cell tumor cell lines. To determine whether exposure to cDDP would select for cells with increased MT expression, the MT content of the human teratocarcinoma cell line T7800 was measured after development of resistance to cDDP by exposure to progressively higher drug concentrations (6.25-25 microM). cDDP-resistant cells (T7800R) had significantly higher MT mRNA and MT protein, increased resistance to killing by cDDP and altered in vitro growth kinetics compared to parental T7800 cells. cDDP resistance in a variety of other human tumor cell lines correlated with MT content, with no significant difference in glutathione level. These data indicate that selection in vitro for cDDP resistance in human germ cell tumors coselects for cells with enhanced MT content. However, selected cells differed in characteristics other than MT content. They had a slower growth rate and, although the rank order of MT level in T7800, T7800R and other human tumor cell lines correlated very well with cDDP resistance, differences in the level of MT expression did not correspond with differences in the absolute level of cDDP resistance. These results suggest that increased MT expression is concomitant with increased cDDP resistance in a variety of human tumor cell lines. However, measured differences in MT levels may not accurately reflect the degree of cDDP resistance differences among those cells.

Cell Division↗
PubMed1994

Activation of human monocytes with lipopolysaccharide induces metallothionein expression and is diminished by zinc.

The metal-binding protein metallothionein (MT) confers resistance to the toxic effects of metals. Although a role for MT in metal homeostasis and protection against toxic free radicals has been suggested, no clear physiological function has been established. The ability of human monocytes to be activated by bacterial lipopolysaccharide (LPS) treatment provided a model to investigate the effect of zinc on both cellular activation (H2O2 production) and MT expression. In both primary human monocytes and a monocyte-derived cell line (THP-1), LPS induced activation and MT expression; it did not induce MT expression in nonmonocyte human cells. Treatment of THP-1 cells with nontoxic zinc levels increased MT accumulation. Subsequent treatment with LPS resulted in a decrease in both MT mRNA and protein levels and inhibited the ability of THP-1 cells to undergo the respiratory burst. Pretreatment with cadmium had the same inhibitory effect. We conclude that MT expression is associated with monocyte activation, and exposure to zinc or cadmium interferes with the ability of monocytes to respond to activation signals. Metallothionein may play a role in that response.

Base Sequence↗
PubMed1994

Role of metallothionein in carcinogenesis.

Metallothionein (MT) is a low-molecular-weight protein (6800 Da) and one-third of its amino acids are cysteine residues. The 20 cysteines coordinate 7 metal atoms (zinc, copper, and/or cadmium). This protein is extremely inducible by metals as well as a number of organic compounds. MT is though to be an important intracellular storage site for zinc and possibly other essential trace elements. In addition, tolerance to cadmium toxicity is often due to the induction of MT, which sequesters cadmium and lowers its concentration at critical intracellular sites. Recently it has been proposed that MT might play important roles in several aspects of the carcinogenic process. In this context a symposium was held recently on this topic at the 1993 Annual Society of Toxicology Meeting. At this symposium Dr. Cherian discussed the expression of MT in various human tumors and its use as a potential marker of tumor differentiation or cell proliferation. Dr. Imura provided data illustrating that induction of MT can be used as an adjunct in cancer chemotherapy, in preventing toxicity caused by gamma-irradiation or cisplatin (CDDP) and other chemotherapeutics. Induction of MT has been suggested to be an important mechanism of resistance of tumor cells to chemotherapeutic agents, such as CDDP. This is controversial, and various views on this topic were presented by Drs. Howell, Lazo, and Koropatnick. Dr. Waalkes then discussed the role of MT in the carcinogenic and anticarcinogenic effects of metals.

Animals↗
PubMed1994

Antisense down-regulation of metallothionein in a human monocytic cell line alters adherence, invasion, and the respiratory burst.

A human monocyte-derived cell line (THP-1) was used as a model to investigate the role of metallothionein (MT) in the cellular physiology of resting and activated monocytes. MT protein levels were reduced in THP-1 cells by transient transfections with an antisense MT expression vector. Antisense mouse MT-1 RNA was constitutively expressed under the control of the H-2Kb (mouse major histocompatibility complex I) promoter and could be further induced by lipopolysaccharide (LPS) treatment. THP-1 cells expressing antisense MT RNA (aMT-THP-1) had a 30% reduction in MT protein levels. In the absence of LPS treatment, aMT-THP-1 cells demonstrated increased production of H2O2 concurrent with enhanced adherence and invasiveness compared to cells transfected with the control vector (cv-THP-1). Treatment of aMT-THP-1 cells with LPS depressed these activation-associated responses and further reduced the level of MT protein. cv-THP-1 cells activated by LPS produced high levels of H2O2 and adhered to and invaded a reconstituted basement membrane. In addition to increasing cadmium sensitivity, diminished MT levels affected broad-ranging processes associated with resting and activated monocyte function. Thus, metallothionein plays an important physiological role in cells in addition to its role in detoxification of heavy metals.

Base Sequence↗
PubMed1993

A mutant mouse (tx) with increased hepatic metallothionein stability and accumulation.

Metallothioneins (MTs) are low-molecular-mass cysteine-rich proteins implicated in metal homoeostasis and resistance to toxicity induced by heavy metals and alkylating agents. We report high hepatic MT protein accumulation (greater than 100-fold compared with wild-type mice) in toxic milk (tx) mice, along with markedly higher cytosol copper and zinc levels. Increased MT-gene transcription alone could not account for the high constitutive MT protein levels, since MT mRNA levels were not increased in tx mouse livers. However, hepatic MT was significantly more stable in adult tx mice: MT half-life (t1/2) was 79 or 77% greater than in wild-type mice before and after Cd induction respectively. Cd or Zn treatment increased MT mRNA, but not MT protein, accumulation in tx mouse livers: Cd displaced MT-bound Zn and Cu in preexisting MT. Thus tx mice appear to accumulate hepatic MT as a result of decreased protein degradation. These animals may provide a useful model to study the physiological role of MT, and human diseases (such as Wilson's disease) with abnormal copper metabolism.

Animals↗
PubMed1993

Altered cisplatin and cadmium resistance and cell survival in Chinese hamster ovary cells expressing mouse metallothionein.

Metallothionein (MT) proteins are associated with resistance to the toxic effects of heavy metals, chemotherapeutic drugs, and alkylating agents. It has been suggested that MT may mediate both resistance to toxic agents and cellular metal homeostasis. To study the role of MT, we obtained cells expressing a range of MT levels in the absence of heavy metal induction. We cotransfected the eukaryotic G418 resistance vector pSV2neo and mouse MT-1 cDNA in a pBR322 vector into Chinese hamster ovary cells. Of 200 transfected clonal cell populations, five had constitutive MT expression ranging from 31 to 87 ng of MT/mg of protein. All five populations had increased resistance to cadmium but were less resistant to cisplatin than control cells. On the other hand, the level of foreign MT expression correlated well with the degree of cisplatin resistance among the five clones. Resistance to ionizing radiation and growth rate in the absence of drug or radiation treatment were not affected. However, transfected MT gene expression inhibited the ability of Chinese hamster ovary cells to form colonies in the absence of toxic drug treatment (r = -0.95). The perturbation of cisplatin sensitivity after genetic alteration of MT expression indicates a role for MT in drug resistance: however, the fact that transfected MT gene expression decreased rather than increased drug resistance and decreased plating efficiency in the absence of drug implies that the role of MT may not be one of simply "scavenging" toxic molecules. These data suggest a role for MT in homeostatic cellular processes that, when distributed by transfection of active MT genes, have an effect on cellular drug resistance.

Animals↗
PubMed1993

Production of a bifunctional hybrid molecule B72.3/metallothionein-1 by protein engineering.

A hybrid anti-tumour B72.3 antibody/metallothionein protein B72.3MT-1 was produced by the construction of the expression vector mpSV2neo-EP1-B72.3MT-1. This vector contained the neo gene as a selection marker, the murine immunoglobulin promoter and enhancer, and the hybrid B72.3 heavy chain gene fragment with mouse metallothionein-1 cDNA gene ligated into its CH2 domain. The expression vector was transfected to the heavy chain loss mutant B72.3Mut(K) cell line. The hybrid protein B72.3MT-1 was purified from transfectant supernates using a Protein G column. We showed that the hybrid protein retained the binding reactivity for the TAG72 antigen as the original B72.3 antibody, and the metal-binding capacity of the native metallothionein molecule. Therefore, the bifunctional hybrid protein B72.3MT-1 may be very useful in cancer imaging when labelled with radionuclides such as 99mTc.

Adenocarcinoma↗
PubMed1992

Arsenic induces and enhances rat hepatic metallothionein production in vivo.

Metallothionein genes (MT) are inducible by a variety of agents, including heavy metals. We report the induction of MT expression by arsenite (As3+) in rat liver in vivo. As3+ (but not arsenate [As5+]) injection increased MT protein and MT-1 and MT-2 mRNA accumulation in liver only, but not in kidney or pancreas. In addition, As3+ enhanced zinc-induced MT protein accumulation in liver without any increase in MT mRNA levels. These data indicate that arsenic may increase MT expression either directly (by inducing MT mRNA accumulation), or indirectly by altering post-transcriptional events. This constitutes an unusual mechanism of enhancement of MT gene expression and appears to be mediated by processes not specifically associated with binding of arsenite to MT in vivo.

Animals↗
PubMed1991

Radioimmunoassay of metallothionein in rabbit, rat, mouse, Chinese hamster, and human cells.

We describe a competitive, solid-phase radioimmunoassay for metallothionein, which employs a rabbit antiserum directed against rat MT-2 to detect metallothionein (MT) from several different species (rabbit, mouse, rat, Chinese hamster, and human). The lower limit of detection of the assay for rat MT-2 was 0.7 ng; for rabbit MT-2 it was 2 ng. The method is capable of measuring both isoforms of MT (MT-1 and MT-2). When MT levels in rat and mouse tissues were estimated with this RIA and the silver-saturation method, both assays gave the same pattern of MT induction in control and cadmium-treated animals. Both methods measured high levels of MT in human liver samples. Chinese hamster ovary cells induced with cadmium also showed elevated MT expression. The detectability of MTs from a broad range of species is facilitated by the use of solid-phase MT, which has an avidity for the antiserum similar to that of the MT in the tested sample.

Animals↗