EP0946192A1 - Utilisations de thioredoxine - Google Patents

Utilisations de thioredoxine

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Publication number
EP0946192A1
EP0946192A1 EP97952292A EP97952292A EP0946192A1 EP 0946192 A1 EP0946192 A1 EP 0946192A1 EP 97952292 A EP97952292 A EP 97952292A EP 97952292 A EP97952292 A EP 97952292A EP 0946192 A1 EP0946192 A1 EP 0946192A1
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Prior art keywords
thioredoxin
cells
trx
human
cell
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German (de)
English (en)
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Garth Powis
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Individual
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention generally relates to the use of thioredoxin as, inter alia,
  • a cell growth stimulator as well as a screen for agents that are useful in reducing or
  • Thioredoxin is a low molecular weight (M r 11,000 - 12,000) redox protein
  • thioredoxin reductase Human thioredoxin, which has 5 cysteine (Cys)
  • thioredoxin contains 3 additional Cys residues not found in bacterial thioredoxin that give it
  • cysteine residues that undergo reversible oxidation to cystine.
  • Cys92, Cys69 and Cys73 are found in mammalian but not in bacterial thioredoxins. Cys73
  • Thioredoxin reduces a variety of intracellular proteins including
  • thioredoxin has remarkable extracellular cell growth stimulating properties. It has been
  • Thioredoxin was first studied for its ability to act as reducing co-factor for
  • Transcription factors regulated by thioredoxin include NF- ⁇ B
  • thioredoxin can regulate AP-1
  • eosinophil cytotoxicity stimulating factor protein called eosinophil cytotoxicity stimulating factor (Silberstein DS, et al., J. Biol Chem
  • ADF adult T-cell leukemia-derived factor
  • ADF has been reported to be secreted by virally transformed
  • Thioredoxin mRNA has been found to be over expressed by some human
  • thioredoxin protein was present in certain human tumor cells, and it has not been known that
  • thioredoxin protein played any role in preventing or enhancing tumor cell growth. While thioredoxin itself is known, its use in identifying agents that inhibit cell
  • Human thioredoxin reductase has been characterized as a protein (Oblong JE,
  • MDS myelodysplastic syndromes
  • leukemia may occur in one-third of the patients.
  • the underlying defect is decreased
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • IL-3 displays multilineage progenitor stimulatory effects in normal marrow clinical
  • the present invention relates to the use of thioredoxin as, mter alia, a cell
  • tumor cell growth in a tumor cell that over-expresses thioredoxin comprising contacting said
  • Such agents can include, ter alia, small molecular compounds that complex
  • Such agents can include, inter alia, antibodies to this redoxin, compounds that inhibit the
  • thioredoxin comprising measuring thioredoxin expression or activity in a first sample of said
  • the present invention is based, at least in part, on the discovery that
  • thioredoxin protein is over-expressed in certain human tumor cells; that thioredoxin stimulates the growth of cancer cells; that thioredoxin inhibits apoptosis; that thioredoxin is
  • the present invention involves the new uses of thioredoxin, thioredoxin
  • reductase and mutant forms of thioredoxin for use in screening for anti-tumor agents. It has
  • anti-thioredoxin and/or anti-thioredoxin reductase agents for use as anti-tumor
  • the present invention further relates to the use of thioredoxin and/or
  • thioredoxin reductase antibodies for use as anti-tumor agents.
  • the present invention further relates to the use of anti-sense thioredoxin or
  • anti-sense thioredoxin reductase compounds for use as anti-tumor agents.
  • the present invention further relates to the use of thioredoxin nucleic acid
  • probes and/or thioredoxin antibodies in a diagnostic assay for certain cancers.
  • the present invention further relates to the use of thioredoxin as a target for
  • agents to be used in combination with existing and new treatment therapies such as drugs
  • tumor cells or to increase the sensitivity of tumor cells to these modalities.
  • mutant forms of thioredoxin provide proteins with additional
  • Fig. 1 shows a chart that illustrates the stimulation of human bone marrow
  • CFU-GEMM multilineage progenitors
  • BFU-E erythroid progenitors
  • V V
  • CFU-GM myeloid progenitors
  • Fig. 2 shows a chart that illustrates potentiation of IL-2 induced MCF-7 breast
  • Fig. 3 shows a chart that illustrates the inhibition of thioredoxin stimulated
  • Figs. 4A-B illustrate comparative charts showing the effects of thioredoxin
  • Nucleotide means a monomeric unit of DNA or RNA consisting of a sugar
  • nucleoside base and sugar is called a "nucleoside".
  • the base characterizes the nucleotide.
  • DNA bases are adenine ("A”), guanine (“G”), cytosine (“C”), and thymine (“T”).
  • A adenine
  • G guanine
  • C cytosine
  • T thymine
  • uracil substitutes for T.
  • an A on one strand pairs with
  • DNA comprises deoxyribose as the
  • RNA comprises ribose
  • amino acids are shown either by a three letter or one letter abbreviation as
  • DNA Sequence means a linear array of nucleotides connected one to the
  • Codon means a DNA sequence of three nucleotides (a triplet) which
  • ATG is a translation start signal.
  • Proteins are composed of a linear array of amino acids
  • Gene means the entire DNA of an organism, cell or a virus. It includes,
  • Gene means a DNA sequence which encodes through its template
  • mRNA messenger RNA
  • cDNA means a complementary or copy DNA prepared by using mRNA as a
  • oligonucleotide primer and a mixture of nucleotides.
  • PCR means a polymerase chain reaction whereby a specific DNA sequence, either genomic or cDNA, can be preferentially amplified by the enzyme Taq polymerase
  • Transcription means the process of producing mRNA from a gene or DNA
  • Translation means the process of producing a polypeptide from mRNA.
  • “Expression” means the process undergone by a gene or DNA sequence to
  • Plasmid or phagemid means a nonchromosomal double-stranded DNA
  • a plasmid may be changed or transformed as a result of the DNA of the plasmid.
  • a plasmid may be changed or transformed as a result of the DNA of the plasmid.
  • plasmid carrying the gene for ampicillin resistance transforms a cell previously
  • a cell transformed by a plasmid is
  • Recombinant DNA Molecule or “Hybrid DNA” means a molecule
  • Apoptosis is programmed cell death activated by a genetic program to
  • Oncogene is a gene that encodes a protein able to transform cells in culture to induce cancer in animals.
  • FBS Fetal bovine serum
  • NIH 3T3 cells In a non-limiting embodiment of the present invention, NIH 3T3 cells
  • a nuclear localization signal causes malignant transformation of the cells.
  • lymphoid cells with human thioredoxin DNA has been shown to inhibit apoptosis induced by
  • glucocorticoid N-acetylsphingosine, staurosporine, thapsigargin
  • the thioredoxin gene acts as an oncogene according to the
  • an oncogene a gene that encodes a protein able to transform cells in
  • the thioredoxin gene offers an increased
  • the cells differentiate and move down the villi to eventually be shed into the gastric lumen
  • thioredoxin levels decrease.
  • human MCF-7 breast cancer cells with cDNA for human wild-type thioredoxin or with
  • thioredoxin increases the density to which the NIH 3T3 cells grow in culture and stimulates anchorage-independent colony formation by MCF-7 breast cancer cells.
  • the redox-inactive thioredoxin increases the density to which the NIH 3T3 cells grow in culture and stimulates anchorage-independent colony formation by MCF-7 breast cancer cells.
  • mutant thioredoxin acted in a dominant-negative manner, so that transfected MCF-7 cells
  • embryonic cells with human thioredoxin cDNA increases their growth rate and cell saturation
  • the cells are inoculated into immunodeficient (scid) mice.
  • Trx was originally studied for its ability to act as a cofactor for ribonucleotide reductase. the first unique step in DNA synthesis (1). Human Trx was subsequently shown to modulate the DNA binding of several transcription factors that regulate cell proliferation, including nuclear factor KB ( 2). the glucocorticoid receptor (3), and. indirectly through the nuclear redox protein ReM . activator protein- 1 (Fos Jun heterodimer. Ref. 4). Cloning and sequencing of human Trx have shown that it has a predicted amino acid sequence (5. 61 identical to that of a growth factor secreted by virus-transformed leukemic cell lines, termed adult T-cell leukemia-derived factor (7. 8). Human Trx. but not bacterial Trx. added to the culture medium stimulates the growth of a variety of normal and cancer cell lines (8-10). The addec Trx is not taken up by cells ( 1 1 ) and appears to stimulate cell growtr
  • Trx is required for growth stimulation, and redox-inactive mutant Trxs do not stimulate cell growth ( 10 ) .
  • Trx mRNA levels are increased compared with corresponding normal tissue in almost half human pnmar ⁇ lung ( 5 ) and colon tumors examined ( 13).
  • Trx protein has been reported to be increased in human cervical neopiastic squamous epithelial cells ( 14) and hepato- cellular carcinoma ( 15 ).
  • Trx is excreted from cells ( 16- 18) using a leaderless secretory pathway ( 17). and we have suggested that Trx might be a growth factor for some human cancers ( 1 1 ). However, it remains to be unequi ocally demonstrated that endogenously produced Trx can affect cell proliferation. The role Trx plays in the transformed phenotype of cancer cells also is not known.
  • NIH 3T3 cells Human MCF-7 breast cancer cells and munne NIH 3T3 cells were obtained from the American Tissue Type Collection iRockville. MD). maintained in DMEM containing I0 ⁇ % FBS under 6 c CO : at 37 * C. and passaged before confluence.
  • NIH 3T3 cells were transfected wuh Trx:pRXneo. Trx. pDC30 neo. C32S/C35S.pDC304neo. or pRXneo alone.
  • MCF-7 cells were transfected with Trx:pDC304neo. C32S/C35S:pDC304neo. or pDC3Wneo alone.
  • Transfection used liposomes of .V-(l ⁇ .3-dioleolyl)propyl
  • Cells were selected by growing for 4 weeks in DMEM with lOt FBS and 400 ⁇ g/ml G4I8 sulfate ( Life Technologies. Guthersburg. MD). Cell colonies were isolated by ir simzati ⁇ n onto small squares of sienle filter paper and expanded by growing in (he same medium. All studies were conducted on clonal cell lines between passages 3 and 10.
  • Fig. 2 Effects of transfection with Trx or C32S C35S cDNA on the growth of NIH 3T3 cells.
  • Cells were plated in platuc dishes at a density of 2 x 10* cells cnv in DMEM with lOf' FBS and cell number measured daily.
  • NeoC vector atone-nnsfectcd cells.
  • the apparent decrease in the number of ceils after day 3 is due to detachment of ceils from the plastic surface: T. ⁇ . and ⁇ i. Thioo. Thr ⁇ AD. and Trao9 ceils transfected with Trx cONA: D and ⁇ .
  • Tumor tormati ⁇ n b ⁇ transiected NIH 3T3 cells as studied bs the s.c injection of 10' transected cells in 0 l ml of ste ⁇ le 09'r N ⁇ CI into the backs of groups ol tour male SCID mice or itx nude mice.
  • Tumor fom tiun bv MCF-7 cells was studied b> injecting : ⁇ 10' cells in 0.1 ml ol stenle Q.9 ⁇ > NaO and 0.1 ml of M tngel tBecton Dickinson. Bedford. MAi s.c. into the backs of groups of four tem ⁇ le SCID mice that had been implanted s.c.
  • Stausucal analysis was by Student s nonpaired r test unless otherwise stated. Tumor growth rates in SCID mice were linearized using the cube root of the tumor volume by day for each mouse, and ANOVA was performed using Dunnett's test to determine significant differences from the vector alone- transfected (control) cell line.
  • transfected Trx mRNAs probably because the transfected Trx mRNAs also contain portions of the vector promoter region, the 5' leader sequence, or the polyadenylate tail.
  • the level of transfected Trx mRNA expression was relatively low. being only 0.2- 1.4-fold the endogenous mouse Trx mRNA.
  • Western blomng showed no significant increase in the level of Trx protein tn the cells compared with wild-type or vector aione-transfected cells (results not shown).
  • Trx-transfecied NIH 3T3 cells grew at the same rate on a plastic surface, but reached saturation densiues up to twice that of the
  • vector aione-transfected NIH 3T3 cells (Fig. 2).
  • the vector aione- transfected cells had the same growth characie ⁇ sucs of * ⁇ ld-rype NIH 3T3 cells.
  • NIH 3T3 cells transfected with the redox-inacuve C32S/ C35S Trx grew more slowly and reached a lower saturation density on a plastic surface than the vector alone-iransfected cells Neither the vector aione-transfected NIH 3T3 cells nor the Trx or C32S/C35S Trx transfected cells formed colonies in soft agarose (results not shown ) .
  • Trx-transfected NIH 3T3 cells The ability of transfected NIH 3T3 cells to form tumors when inoculated into tmmunodeficiem mice is used to identify neopias ⁇ c transforming genes (24).
  • Trx-transfected NIH 3T3 cells Thio ⁇ or ThioAD were injected i.m. into SCID or nude mice, there was no tumor formation over 40 days (results not shov.nl. Thus. Trx expression, at least at the level obtained in this study, was not. by itself, transforming.
  • Trx and C32/C35S Transfection of MCF-7 Breast Cancer Cells Human solid cancer cells generally show a greater proliferation response to added Trx than do mouse fibroblasts ( 10. 1 1). This is shown for MCF-7 human breast cancer ceils compared with NIH 3T3 cells in Fig. 3. Thus, we also studied the effects of Trx transfection using MCF-7 breast cancer cells. Transfection of MCF-7 cells with Trx: pDC304neo yielded 31 clones that stably overexpressed Trx mRNA. and transfection with C32S/C35S.pDC304neo yielded 45 clones stably expressing C32S/C35S mRNA.
  • Fig. 4 Expression of transfected mRNAs by some of the clones is shown in Fig. 4 As seen previously with the mouse cells, the transfected human Trx mRNAs tn MCF-7 cells were larger than endogenous human Trx mRNA. The level of Trx mRNA expression was up to 0 8-fold and C32S/C35S mRN up to 2 1 -told the endogenous Trx mRNA levels. Light microscopy showed no difference m the appearance of vector aione-transfected and Trx- transfected MCF-7 cells growing on glass overslips ( Fig ⁇ . and both were similar to wild-type MCF-7 cells In contrast. C32S/C35S- transfected MCF-7 cells appeared more rounded and had ⁇ reduced cytoplasm-to-nucleus ratio
  • Trx-transfected MCF-7 cells showed linear growth characteristics on plastic surfaces over 7 days.
  • the Trx-transfected cells grew at the same rate as the vector aione-transfected MCF-7 cells (Fig. 6). However, when grown in the absence of. or with 0.5 ⁇ t FBS for 2 days, the Trx-transfected cells grew at twice the rate of the vector aione-transfected cells (results not shown).
  • the C32S/C35S-trans- fected cells grew at a significantly slower rate that was 56-78 ⁇ of the Fit 5 lifht microicops ol Trx wdCj'.S/C.'JS cDN'A-tniuiecied MCF-7 breau cancer cell!.
  • the cells were crown to 731 conlluet e on
  • Tissues from the injection site and other organs were taken for histologies! examination at the end of the study.
  • the animals injected with vector alone or Trx-transfected cells showed large solid tumors.
  • the animals injected with Q-2 C33S-transfeeted cells showed small microscopic rumor cell deposits. There was no evidence of tumor metastasis to other organs in any of the animals.
  • Northern analysis of the tumor taken from animals injected with Trx-transfected cells showed the presence of transfected Tn mRNA as determined by its large size ⁇ results not shown i.
  • Trx regulates the redox state and activity of a rtumoer of intracellular proteins that control cell irowth. including ⁇ oonucleoude reductase ( I) and the DNA binding of several transcnotion factors (2— i ⁇ Recombinant human Trx added to normal and cancer ceils in culture stimulates their proliferation 11 1 ).
  • ⁇ oonucleoude reductase I
  • 2 i ⁇
  • Trx i ⁇ Recombinant human Trx added to normal and cancer ceils in culture stimulates their proliferation 11 1
  • Trx may play tn malignant transformation of cells is not known. The present study was undertaken to address some of these questions.
  • NIH 3T3 ceils transfected with T ⁇ t showed an increased cell saturation density when grown as a monolayer on plastic surfaces. Loss of contact inhibition is a feature of transformed cells (24).
  • Trx-transfected NIH 3T3 cells did not form rumors when inoculated into tmmunod- eticient mice.
  • the Trx-transfected MCF-7 ceils did not show increased growth on plastic surfaces in normal FBS. but exhibited significantly increased anchorage-independent growth measured by colony formation in soft agarose. It is surprising that when the Trx-transfected MCF-7 cells were grown as xeno rafts in SCID mice, they exhibited decreased growth rate compared with vector aione- transfected MCF-7 cells.
  • Trx can stimulate the immune system of mice' so that T ⁇ t secreted by the transfected MCF-7 celts might promote some immune rejection, even by the SCID mice, which, although deficient in mature B and T lymphocytes, have natural killer-, myeioid-. and antigen-presenting ceils ( 26 ⁇ .
  • SCID mice which, although deficient in mature B and T lymphocytes, have natural killer-, myeioid-. and antigen-presenting ceils ( 26 ⁇ .
  • Both NIH 3T3 and MCF-7 breast cancer cells transfected with the C32S C23S Trx showed slowed growth rates on a plastic surface.
  • colony formation by MCF-7 breast cancer cells in soft agarose was considerably decreased.
  • the C32S/C33S- ⁇ ransfected MCF-7 cells formed only microscopic tumors.
  • C32S C33S is a rcdoi-inactive mutant Trx that acts as a competitive inhibitor of T ⁇ reductase ( 10).
  • Trx acts as a competitive inhibitor of T ⁇ reductase ( 10).
  • Our X-rav crystallographic studies have identified a highly conserved 1. mmo acid hydrophobic surface on mammalian, out not bacterial. Tr ⁇ ». whicn t- ⁇
  • C31S/C35S is very similar to that of Trx (27) so that C32S/C3SS is likely to participate in the formauon of a heterodimer with Trx and thus might lower Trx monomer concentrations or affect the biological activity of the dimer.
  • C32S/C35S does not stimulate cell growth when added to the culture medium.
  • C32S/C35S might also act as a competitive inhibitor to the normal redox-acnve substrates of Trx Whatever the mechanism, it appears that C32S/C35S acts in a dominant-negative manner to inhibit the effects of endogenous Tn and. in so doing, inhibits cell growth and reverses the transformed phenotype of MCF-7 breast cancer cells.
  • Trx is known to be secreted from cells by a Ieaderless secretory pathway ( 17). The concentrations of Trx found in the medium, up to 10 nvt after 6 h. are lower than those required to directly stimulate cell proliferation ( I I ).
  • Trx at nanomoiar concentrations will potentiate the growth effects of cytokines such as ⁇ nterieuk ⁇ n-2 and basic fibroblast growth factor 4 . It remains to be established whether the extra Trx is producing its effects on cell proliferation through an intracellular or an extracellular action.
  • Trx binds to the urface of cells ( 1 1. 28) so that secreted Trx could have a local effect at the outer cell surface although concentrations tn the medium are low
  • the levels of transfected Tn mRNA in cells were not high, only up to 1-fold endogenous Trx mRNA levels, and were independent of the mammalian transfection vector used Typically. mRNA levels resulting from transfection using such vectors are 10— 50-fold or higher (29). It may be that high levels of Trx gene expression are toxic to cells. We have found only a low expression of the human Tn gene in ⁇
  • transge ⁇ ic mice 4 In .oniraM, some human tumors show very hi-j levels ⁇ i Tn mRN ⁇ compared with the normal tissue: mure than 1 1 -told in human p ⁇ murv lung tumors i 30) and even higher in human p ⁇ mars ol n tumors ⁇
  • thioredoxin was over-expressed in tumor cells compared to normal mucosa, and in all cases
  • lymphocytes Levels of thioredoxin significantly higher than in normal dividing cells, were
  • nuclear proliferation antigen was detected by Ki67 antibody and
  • TUNEL in situ terminal deoxynucleotidyl transferase
  • thioredoxin protein is directly associated with highly proliferative tumors.
  • MCF-7 human breast cancer cells were transfected with cDNA for thioredoxin
  • thioredoxin protein production measured by quantitative Western blotting, up to 100% that
  • thioredoxin can reverse the transformed phenotype and inhibits tumor growth in vivo
  • thioredoxin is a known protein, it has not been disclosed or
  • thioredoxin causes inhibition of anchorage-independent growth of the cells in soft agarose
  • the redox-inactive mutant is
  • agents may be antibodies, drugs or antisense.
  • imidazolyl disulfides inhibit thioredoxin-dependent cell growth (Oblong JE, et al., Cancer
  • NCI was conducted in order to identify compounds with a similar pattern of growth
  • thioredoxin reductase is the only known way for thioredoxin to be
  • reductase could also be a target for the development of anti-cancer drugs.
  • modified thioredoxin does not undergo spontaneous oxidation and/or
  • dimer formation or protected against breakdown by blood and tissues, may have therapeutic
  • transgenic animals in need of stimulation of body growth; (7) in need of simulation of the
  • the underlying defect in myelodysplastic syndrome is decreased multilineage
  • progenitor cell growth associated with decreased sensitivity to growth factor stimulation.
  • Thioredoxin acts to increase the sensitivity of cells to growth factors and stimulates
  • multilineage progenitor cells which provides a beneficial utility in individuals with MDS.
  • Thioredoxin may be used to protect
  • Wild-type and Cys73 ⁇ Ser mutant thioredoxin also stimulates the growth of
  • fibroblasts which are important components of wound healing process.
  • transgenes with wild-type or mutant forms of thioredoxin, with or without tissue specific
  • inducible promotors could be used to stimulate the development of the animal or the
  • cytokines such as IL-2 and fibroblast growth factor (FGF).
  • IL-2 factors given directly or themselves as gene therapy, for example IL-2.
  • modified thioredoxin also stimulates cell growth.
  • thioredoxin has a highly conserved hydrophobic dimer forming surface and that Cys73
  • Cys73 ⁇ Ser thioredoxin is as effective as wild-type thioredoxin at stimulating cell
  • Cys73 ⁇ Ser mutant thioredoxin can prevent the death of lethally y-irradiated mice.
  • mice There were 6 mice in the control group and 4 mice in the treated group. The
  • Cys73 ⁇ Ser mutant thioredoxin stimulates colony formation by the muitilineage progenitor
  • CFU-GEMM but does not stimulate the lineage specific erythroid progenitor (BFU-E)
  • CFU-GM myeloid progenitor
  • Fig. 1. illustrates the stimulation of human bone marrow colony formation by
  • Cys73 ⁇ Ser mutant thioredoxin in accordance with the present invention.
  • Human bone marrow was obtained as excess material from normal allogeneic bone marrow donors.
  • CFU-GEMM CFU-GEMM
  • BFU-E erythroid progenitors
  • V myeloid progenitors
  • IL-2 interleukin-2
  • FGF fibroblast growth factor
  • Fig. 2 illustrates potentiation of IL-2 induced MCF-7 breast cancer cell growth by
  • Cys73 ⁇ Ser mutant thioredoxin in accordance with the present invention. Cells were growth
  • antibodies to the receptors for the growth factors can block the
  • thioredoxin 1 ⁇ M; monoclonal antibodies to FGF receptor, IL-2-receptor and
  • the EGF and EGFR were added as a negative
  • modified thioredoxin does not undergo spontaneous oxidation and/or dimer formation has a
  • proteolytic cleavage to prevent breakdown by plasma enzymes.
  • Thioredoxin/mutant thioredoxin may have use after bone marrow
  • thioredoxin might be more effective in this regard.
  • the use of mutant thioredoxins may not be
  • Thioredoxin is a wiJelv disrnruted redox protein ch t reyulate* sever.ii intracelluiar redov - "cea.e. and stimulates the proliferation of both normal and tumor cells. Wc have round :hat when ihsence of reducing auents. human recombinant Trx undergoes spontaneous oxidation, iosin it ' uinre ;ell yjrowth. bur is still a substrate tor NADPH-derendenr reduction human thioredoxin :re '..
  • Trx ro a that ⁇ > nor a su strat r ' or • eduction T reductase and thar does not stimulate cell proliferation. Both conversions can be induced .ancs and are reversible bv treatment with the chiol reducins auent dithiothrettol. STS- PAGE Trx undernoes oxidation to monome ⁇ c form,s> precedin ⁇ dimer formation.
  • Trx forms a Jtmer that is stahilt.ed by an tntermolecular Cvs '-Cvs .
  • C73S C73S was prepared to determine the tele of Cys' 1 in oxidative rowth stimulation.
  • C73S was as effective a» Trx in s ⁇ mula ⁇ ns cell growth and was a comparable iioredoxin reductase.
  • C73S did not sh ⁇ w spontaneous or oxidant-induced loss of acnvttv and did ner.
  • the results suggest that Trx can exist in monome ⁇ c forms, some of which are mediated bv not stimulate cell proliferation but can be reduced by thioredoxin reductase.
  • Cys' is also involved of an en:ymattcally inactive homodimer. which occurs on long term storage or by chemical us. although clearly involved m protein inactivation.
  • Cvs 1 ' is not necessarv for the zrowth ttvitv of Trx.
  • Trx is a redox protein found in both eukaryotes and pro- aryotes [1J.
  • the redox activity of Trx arises from a highly conserved T -Cys-Gly-Pro-Cys-Lys active site sequence where the 2 cysteine residues (Cys) undergo reversible oxidation to cystine.
  • Reduction of Trx is cataly_ «d by thiore ⁇ doxin reductase (2J.
  • Trx was originally identified in EscJi- ai ia coU as a hydrogen donor for ribonucleotide reductase [3J.
  • Trx has since been found to act as an intracellular dithiol-disulfide reductase and to modulate the activity of a number of intracellular proteins (4-61 including the DNA binding of transcription factors [7-10J. Trx-like sequences are found in other proteins including protein disulfide is ⁇ m- erase (111. There is evidence that Trx may play a role in the growth and transformed phenotype ot some cancers. Trx is
  • Trx act* .w ⁇ ⁇ - enously as a redo -active growth factor.
  • Trx * » • act by a helper mechan ⁇ >m that - ⁇ n « ⁇ t ⁇ :e » the cell? inrowth factors secreted bv the cell.- c> 11 H- Mutant human Trxs.
  • ana could be .tctinu as an autocnne factor tor tne umwth ut Mime cancer ells 1131-
  • T ⁇ t can also orm a homodimer with a 1100 ⁇ 2 inter ace domain and a disulfide bund between Cys 71 from each monomer (20).
  • Cys' - ⁇ Ser mutant human Trx (C73S) was prepared from single-stranded, sense strand human Trx cDNA ligated by polvethylene glycol precipua- non into the pBluescnpt KS vector (Stiatagene. La Jolla, CA) using R408 helper phage.
  • the single-stranded cDNA was used fur uligonucleoride-dtrected m vtrro mu ⁇ genesis (Version 2.1 Kit. Amersham. Biickinghamshirc.
  • the Tn solution was kept at 4* and used within 2 hr (fresh) or stmed in water or 0.1 M potassium phosphate-buttered 0.9% NaQ at 4* or -20* tor specified times.
  • Oxidised Trx for cell trrowth studies was prepared by addinu a 5-told molar excess ⁇ H ; 0» to a 25 ⁇ M Trx stock solution without DTT and I hr later removtnc unreacted H : 0 ; ustnu a PD-10 column.
  • MCF-7 human hre.tst cancer cells were obtained from the
  • Trx reduced/min/mg protein Human placenta thioredoxin reductase, specific activity 33J ⁇ mol Trx reduced/min/mg protein, was prepared as previously described [23]. Reduction of Trx and C73S by thioredoxin reductase was measured by the oxidation of NADPH at 340 nM with insulin as the final electron acceptor as described by Luthman and Holmgren (21.
  • Trx. mutant C73S or C32S/ C35S Trxs Trxs that had been aged at room temperature for 48 hr, 7 davs, 90 days; or Trxs treated for I hr with I mM diamide, 10 mM DTT, 3 mM 2-merca ⁇ toethanol or 2:1 (v:v) HsO : , was mixed with an equal volume of loading buffer containing 3% SDS. 10% glycerol and 0.1% brom- phenol blue in 0.05 M Tns-HCl, pH 6.8.
  • Cys' ⁇ Ser mutant Trx (C73S) stimulated the proliferation of human MCF-7 breast cancer cells.
  • the EC W for gmwth stimulation bv C73S was 350 nM and the maximum effect was seen at 1 ⁇ M. which is similar to values we have previously- reported tor stimulation t MCF-7 t-ell proliferation bv recombinant numan T ⁇ . [IS].
  • FIG. I Stimulation of MCF-7 breast cancer call growth by fresh and aged Trx and C73S.
  • Also shown for reference is the effect of 10% fetal bovine serum. Each value is the mean of 3 dete ⁇ ninations, and bars are SEM.
  • Trx scored in the presence of bovine catalase at 1 unit/ml did not lose biological activity over a 5-day period (results not shown).
  • C73S was a good substrate for reduction by human ptacen- tal d ⁇ oredoxin reductase with a K of 0.20 ⁇ M and a V m m of 6.3 run ⁇ Vr ⁇ in/ ⁇ g. These values are similar to those we have previously found for fresh Trx, which were a K favor of 0 J3 ⁇ M and a V ⁇ of 5.9 M ⁇ l/mm/ ⁇ g (23).
  • Trx When stored in H : 0 either at -20* or at room temperature Trx showed a loss of activity with a hatf-Ufe of
  • the lower pH of the Hilutum in water could stabilize Trx or the increa s e in ionic strencth of phosphate-buffered 0.9"; NaCl could enhance the formation of the inacnve homodimer oi Trx.
  • the aaed Trx showed a slow, delayed reduction bv thioredoxin reductase that was stimulated by catalytic amounts of fresh Trx (Ftp. 2). It is important to note that the loss at activity of Trx as a substrate for thioredoxin reductase was much slower than the loss of activity as a stimulator of cell growth.
  • C73S did not sh ⁇ w a loss of activity as substrate for thioredoxin reductase upon storaee for up to 30 davs.
  • the ability of Trx to act as a substrate for thioredoxin reductase was completely inhibited by treatment wtrh 5 molar equivalents of H 0 : . whereas C375 remained fullv active after treatment with 100 molar equivalent? oi H : 0 : (Fig. 3).
  • Electrophoretic analysis of freshly prepared human Trx stored in DTT showed a mixture of 5 bands of apparent molecular weights tanging from 6.1 to 11 kDa (Figs. 4, 5, and 6, lane 1 ).
  • Storage of Trx at room temperature without DTT resulted in a change in the banding pattern with disappearance of the 8.1 -kDa band by 48 hr (Fig. 4, lane 2).
  • Storage of Trx without DTT for 7 days resulted in the loss of additional bands and the appearance of a new band at 23 kDa due, apparently, to a Trx dimer (Fig. 4, lane 3).
  • Storage of Trx without DTT for 90 days at 4* resulted un almost complete conversion to the Trx dimer (Fig. 4, lane 4).
  • FIG. 3 The effect of H t O t on the reduction of Trx (filled ban) and C73S (open ban) by thioiedoxin reductase.
  • Trx solutions were treated with varying amounts of H t 0 2 for 18 bra at room temperature.
  • Reductaie activity WH measured by adding treated samples to a solution of 0.1 HEPES buffer, pH 7.6, 5 mM EDTA, 17 ⁇ M insulin, 100 ⁇ M NADPH. 15 ⁇ g/ml human thioredoxin reductase and measuring the race of NADPH oxidation at 340 tun at room temperature.
  • Trx dimer a protein thiol oxidizing agent [251 (Fig. 5, lane 5).
  • the formation of Trx dimer following diamide treatment was also confirmed by gel permeation chromatography (results not shown). H j O; treatment of Trx also caused dimer- izati ⁇ n but produced a different banding pattern to that pnxjuced by diamide (Fig. 5, lane 6).
  • Treatment of Trx with NEM. a thiol alkylating agent [26] gave a single band with a slightiy elevated apparent molecular weight, but no dimer formation (Fit:. 5. lane 4).
  • Trx undergoes at least 2 levels of spontaneous and induced oxidative trans, formation.
  • the first oxidation occurs spontaneously within a few days to a form(s) that can n ⁇ longer stimulate ceil growth but remains a substrate for thioredoxin reductase.
  • the slower oxidation occurs over a period of weeks, or can be induced by the thiol oxidizing agent diamide, and leads to a disulfide bonded homodimer which not only fails to stimulate cell growth but is a poor substrate for thioredoxin reductase.
  • Trx can be induced by chemical oxidation, are protected against by catalase and are reversed by the thiol reducing agent DTT is consistent with the interpretation that the changes in Trx are due to oxidation.
  • Cys 71 appears to play a critical role in both levels of oxidant-induced inactivation since C73S does not lose the biological activity or its ability to act as a substrate for thioredoxin reductase upon aging.
  • Trx fresh human recombinant Trx can exist in at least five different states, which probably reflect the fully reduces state of the protein as well as different intramolecular disulfide bonded states due to the five cysteine residues present in the protein. While the specific nature of these intramolecular disulfide bonds is not known, it is likely that some, at least, are due to non-
  • FIG. 4 Effect of storage on Trx studied by SDS-PAGE. Protein was stained with silver stain. Lane 1, fresh Trx: lane 2, Trx 48 hrs at room temperature without DTT; lane 3. Trx 7 days at room temperature without DTT; and lane 4. Trx stored 90 davs at 4 ⁇ without DTT. Position of molecular on the left. narurai disultide ⁇ on ⁇ -. structure?
  • the number uf free thiuls in fresh Trx was determined to be 4.5 to 4.6/molecule by Ell- man ' » reauent [2S] (data nut shown), indicating that all five cvstemes are in the sulfhydryl form.
  • Treatment of NEM- alkvlated Trx with oxidizing or reducing agents produces no change in the banding pattern (data not shown), which is runner evidence that all 5 sulfhvdrvls have been alkvlated.
  • FIG. 6 Oxidation and reduction of mutant Trxs studied bv SDS-PAGE.
  • Trx may be undergoing "native" mtramolecular disulfide bond formation prior to ele ⁇ rophoresis, which preven ⁇ the formation of random dtsulfide-bond formation seen with denaturation and electrophoresu of fresh Trx.
  • bovine pancreatic trypsin inhibitor [27, 31).
  • FIG. 7 Position of cysteines in human Trx. Ribbons and ball-and-s ⁇ ck representation showing the relative positions of Cys", Cys 11 . Cys", Cys M and Cys", based on the crystal coordinates for the wild type redneed protein (20). None of tne thiols are in a position for rtrnilfirlir bond formation except for the redox active pair Cys 12 and Cys 1 *. The inter- molecular r ⁇ wln ⁇ V bond requiring the least distort-on in the protein would be between Cys 31 and Cy»".
  • Trx homodimer 23-lcDa Trx homodimer. Reducn ⁇ n of the Trx dimer by thioredoxin reductase is slow and delayed, and is stimulated by low concentnin ⁇ ns of fresh Trx, suggesting there may be an autocatalvtic process. A similar conclusion was reached by Ren et al. [34
  • Trx undercoes a ⁇ >ter loss of activity with thioredoxin reductase in PBS versus water indicates that iron-induced oxidation or an increase in ionic strength mav stabilize and enhance dimer formation, which is consistent with the hvdrophohic nature ⁇ i the dimer interface observed in crystals of human Trx
  • Trx is secreted bv cells into the extracellular environment, which is ptedominandv oxidizing, and might be expected to undergo monomenc oxidation. Conside ⁇ ng its ease of formation, it is reasonable to assume that monomenc oxidation will precede oxidative homodimer formation. Whether this might be sufficient to prevent Trx from acting as a growth factor is not known.
  • the formation of the oxidized monomer inside the cell is less likely since it s li can be slowly reduced by thioredoxin reductase and the interior of the ceil is highly reducing.
  • Trx homodimer formation The physiological significance of homodimer formation is also unknown. What might be Trx homodimer has been reported in diamide-treated Jurkat celb [35]. We have observed small amounts of the Trx homodimer bv immuno- bloitmg of untreated MCF-7 breast cancer and other cell lysates (Powis et al., unpublished observations). It is in- tnguing to speculate that formation of an oxidized Trx monomer or homodimer in response to intracellular oxi- dants such as H ; 0 ; might be a wav mammalian cells detect oxidant formation.
  • Trx is believed to exist in normal celb at concentrations from I to 10 ⁇ M [2, 12], though in selected tissues and specific cell compartments this value could be much higher. It is therefore not unreasonable to assume that Trx will undergo homodimer formation in vivo. As we observed with the enhanced inacttvation of Trx tn phosphate buffered saline, we expect dimer formation to precede faster m mvo than we observe m wrro m water. Whether dimer formation m wvo would prevent the taster oxidation to an intramolecular form is unknown. The slow autocatalvtic reduction of the Trx homodimer to the monomer would be a way to restore the cell to normal operating conditions after the induction of oxidative stress.
  • Trx undergoes relatively rapid (over a few days) spontaneous and oxtdant-induced conversion to a form(s) that doe « not stimulate cell proliferation, but is still a substrate for reduction bv thioredoxin reductase. There is much dimer (over a period of weeb) spontaneous oxidation of Trx to a Cvs 7, - rab ⁇ li:ed homodimer form that is not a substrate for thioredoxin reductase and that also does not stimulate cell proliferation. Both conversions can be reversed by treatment with the thiol reducing agent DTT. and K i th appear to involve the Cys 71 residue.
  • the redox protein thioredoxin plays an important role In controlUng cancer cell growth through regulation of DNA synthesis and transcription factor activity. Thioredoxin is overexpressed by a number of human primary cancers and its expression is decreased during dexamethasone- induced apoptosis of mouse WEHT7.2 thymoma ceils. We examined the ability of WEHT7.2 cells stably transfected with human thioredoxin cDNA showing increased levels of cytoplasmic thioredoxin to undergo apoptosis in vitro and in vivo.
  • the cells were protected from apoptosis induced by dexamethasone, staurosporine, etoposide, and tharnigargin, but not by JV-acetyl-sphingosine.
  • the trx-transfected cells formed tumors that showed increased growth compared to wild-type, as well as ⁇ c/-2-transfected, WEH17-2 cells.
  • the trx- and £>c/-2-transfected cell tumors both showed less spontaneous apoptosis than tumors formed by the wild-type ceils.
  • f ⁇ r-translected cell tumors did not show growth inhibition upon treatment with dexamethasone. This study suggests that increased thioredoxin expression in human cancers may result in an increased tumor growth through inhibition of spontaneous apoptosis and a decrease in the sensitivity of the tumor to drug-induced apoptosis.
  • Trx 3 is a low molecular weight redox protein found in both pro- karyotic and eukaryouc cells (1).
  • the cysteine residues at the conserved Cys 32 -Gly-Pro-Cys 35 -Lys active site of Trx undergo reversible oxidauon-reduction catalyzed by the NADPH-dependent selenium- containing flavoprotein Trx reductase (2).
  • Human Trx is a protein of ⁇ f r 1 1.500 with 27% amino acid identity to Eschenchia coli but containing three additional Cys residues not found in bacterial Trx that give the human protein unique biological properties (3).
  • Trx was originally studied for its ability to act as a reducing cofactor for ribonucleotide reductase. the first unique step in DNA synthesis (4). More recently. Trx has been shown to exen redox control over a number of transcription factors, including nuclear factor KB (5), transcription factor I11C (6). BZLF1 (7), and the glucocorticoid receptor (8). and indirectly, through nuclear redox factor Ref-1/HAPE, Trx can regulate AP-1 (Fos/Jun heterodimer; Ref. 9).
  • Trx is also a growth factor with a unique mechanism of action.
  • Human Trx stimulates the proliferation of both normal fibroblasts and a wide va ⁇ ety of human solid and leukemic cancer cell lines (10, 11).
  • Redox activity is essential for growth stimulation by Trx.
  • mutant redox-inactive forms of Trx lacking the active sue Cys 3: and Cys 35 residues are devoid of growth stimulating activity (1 1 ).
  • Studies with ' ⁇ Mabeled Trx have revealed no high-affinity binding sues that
  • Trx thioredoxin scid. severe combined im unodefi- ⁇ rnl CAT chlnramnhenicni acetvltran ferase might suggest receptors for Trx on the surface ctfcaaccr cells (12). Trx appears to -» «"»ni— cell raOliferanon by ittcRating the sensiuviry of the cells to growth factors secreted by the ceUs themselves (12).
  • Trx overexpresaion may be a factor in the growth of some i ⁇ ftff 1 cancers.
  • Trx gene expression is decreased during n>xa ⁇ nethasone-inch ⁇ ced apoptosis of mouse triytnorr-a-derived WEHT7-2 cells (17).
  • Trx cDNA btixnan Trx cDNA
  • fxtminrri the effects on both spontaneous and drug- induced apoptosis in vitro and with the cells growing in scid mice.
  • Trx cDNA Human wild-type Trx cDNA was prepared as described previously, cloned uao the ⁇ forl sne of the pDC-MM ⁇ co mammalian tiw-sfccnon veci ⁇ r ( 16) and transfected by clecsoporsflon uao mouse W ⁇ H ⁇ .2 trjy ⁇ nc*na-derived cells (18).
  • Tunife ⁇ rd cells were maintained at cuhure de-tancs up to 10* ccUs/ml in DMEM romainrnj 10% fetal b ⁇ vme serum supplemented with 800 ⁇ ml C 18 surfate, and clo ⁇ ea were laolased ia soft afarose and then maintained u. 200 uf/ml G418 sulfate.
  • AU studies were nmriurirri on clonal hues between passages 3 and 20.
  • Stably transfected M-2 WEH-7-2 celb (W_Hbl2 cells) were obtained from Dr. Roger Miesfeld (Umversrry of Arizona. Arlington. AZ: Ref. 19).
  • Drup were added at a culture density of I X 10* to 2 X 10 s cxUs ml.
  • Stock solunons (10 mM) of (texam-that ⁇ ne were utepsiui ia ethanci, whereas -taurotporme. etoposide. th- ⁇ st ⁇ rtui. and N-acesyl-sphuigostne were prepared tn DMSO. Further d ⁇ oons were made unng culture medium.
  • Gsacscart-coid BareptTi The level of funcnonal fjucocorucoid receptors was assessed using a transient ccvansfecuoo of cells with a glucocorocoid response elemenf CAT reporter p-tsrmd (p ⁇ unCAT; Ref. 20) and ⁇ -falac ⁇ o- ndase. After a 22-h recovery period, the cells were treated with I ⁇ M dexa- me aaone. and CAT protein was measured after in additional 24 h unng s CAT EL1SA (Boeh ⁇ nger Mannheim. lncJanapolu. IN). An aliquot of the transfecied cells was stained for 0-salsct ⁇ uda-e activity and CAT activity normahzed for transfection efficiency.
  • Apiptnr-i Apoptotu was measured by an E ISA for htsto ⁇ -a-sociated DNA frasmema (21). by morphotofy and by flow cytometry (22).
  • the cntena uaad for the ⁇ >orphc of ical idennficauon of apoptonc ceils included condensation and rr-crfinsoon of the chromc ⁇ n with the formation of crescents, cellitn aie. increased -uinint. nuclear frsfToentanon.
  • the coverslips were exposed lo a I 1 (X) dilution ot goat aniirabbit biotinsiaied IgC for I h. washed with PBS. exposed to a 1.30 dilution ot fluorescein streptavidm fluorochrome. and again washed with PBS Cells were examined using a Leica TCS-ID laser scanning contocai microscope with an excitation wavelength of 488 nm. For subcellular localization studies ut Trx. Cy5 i indodicarbocyanme) streptavidm was used as the fluorochrome.
  • Tumor lormation by wild-type and iranstected WEHI7 2 cells was studied bv injecting 2 x 10' ceils in 0.1 ml ot mat ⁇ gel s.c into the flanks ol groups ot 20 temale scid mice. Tumor volume was measured with calipers, and mice were euthanized when the tumor volume exceeded 2 cm"
  • 10 mice trom each group were injected i p with I mg/kgdav dexamethasone in lO ⁇ r ethanol in 0 T- NaCI Control mice were injected with vehicle alone. On dav 14. three mice tmm each group were euthanized with CO.. and the tumors were excised and immediately fixed in glutaraldehsde
  • Tissue for Bright-Field Examination The glutaralde- hyde-fixed tissue was posifixed in osmium tetroxide. dehydrated in a graded se ⁇ es ot alcohols and embedded in epoxv resin One- ⁇ m-lhick sections were prepared and stained with loluidine blue for b ⁇ ght-field examination
  • VVEHI7 2 cells were stablv transfected with human Trx cDNA in the pDC304neo mammalian transfection vector
  • Tr ⁇ 5 and Trx6 Fig IA
  • Tr ⁇ 5 and Trx6 Fig IB
  • the rrt-transfected WEHI7.2 cells were resistant to apoptosis induced by 1 ⁇ M dexamethasone as measured by histone-associaied DNA fragmentation (Fig. 2A l or by flow cytometry (Fig 23). Histological examination of the W ⁇ H17.2 cells revealed a classic apoptotic morphology in response to dexamethasone However, only a small fraction of the cells undergo apoptosis at any one time, and they rapidly progress to fragmented cells For this reason, results are expressed as relative apoptosis rather than percentage of apoptotic cells.
  • Glucocorticoid receptor activity measured using a glucocorticoid receptor/CAT reporter plasmid was not decreased in the rrt-transfected cells (results of three studies not shown)
  • Trx is prestm in ine isioplasm and ihe nucleus of wild-type WEHI7 _ cells ⁇ 5> and 7> ⁇ o in transiecied cells toi
  • the cells were treated with 0.01* ethan ⁇ l vehicle ⁇ > or I ⁇ M dexamethasone (Q). md spoptotis was cute ⁇ urad 24 later. Coiwiwu. men of four detcrmina- uoni; ban. SE. •. P ⁇ 0.05 compared to Neo controi. B. apoptosis measured by flow cytometry showi ⁇ f typical lesulu. Ref rani ⁇ l. R2. and ⁇ J of the vanerpraros ate live nonapoptotK. early apoo- lotic. and late apoptotic cellt. respectively. /. pOC3 4neo vector-alone- transfected control cells: 2.
  • the trx- transfected WEHI7.2 celK formed tumors that grew more rapidly than tumors formed b> cither wild-type or bcl-2- transfected WEHI7.2 cells rFig. 3 ⁇ ) Upon histo- > o
  • tumors formed by the vwld-type cells showed fields of apopiouc cells adjacent lo fields ot viable cell , as well as apoptotic cells ad ⁇ uxed with viable-appearing cells (Fig 3fl ⁇
  • the cells undergoing apoptosis exhibited the classic appearance of condensed and argmated chromaun. some in the form of crescents, and j dense cvtoplasm accompanied by vacuolization
  • the rn -transfecied EHI7.2 cell tumors showed minimal numbers ot cells undergoing O 98/24472 £>
  • Trx may be able to directly reduce redox-regulated nuclear transcription factors, such as AP-1 (Fos Jun heterodimer. Ref. 9). If Trx can enter the nucleus, it may not need other nuclear redox factors, such as Ref- 1 /HAP 1. as has been suggested (9).
  • the rrr-tra ⁇ sfected cells were resistant to apoptosis induced by dexamethasone. Trx has been reponed to be necessary for assembly of the glucoconicoid receptor (8). However, glucoconicoid receptor activity was not decreased in the transfected cells, suggesting that the effects of Trx on apoptosis appear to lie downstream of the glucocorticoid receptor.
  • the /nr-iransfected cells also showed resistance to apoptosis induced by siaurosporine. etoposide. ⁇ /-acetyi-sphingosine. and thapsigargin.
  • Exogenously added human Trx has been reported to inhibit apoptosis induced by tumor necrosis factor a in U937 human lymphoma cells (26). However, we found that exogenously added human Trx did not protect WEHI7.2 cells against apoptosis induced by dexamethasone (27). Tumor necrosis factor a and dexamethasone are thought to trigger apoptosis by different signaling pathways. It may also be that exogenous Trx is not taken up by WEHI7.2 cells. We have found that other tumor cells take up Trx poorly, if at all (12). Clearly, an increase in intracellular Trx achieved by transfection of trx in the present study is associated with resistance of the WEHI7.2 cells to apoptosis induced by dexamethasone and other agents.
  • Bcl-2 is believed to exe ⁇ its inhibitory effects upstream of the activation of the cysteine aspanate proteases cascade (caspase) responsible for the final stages of apoptosis (28).
  • caspase cysteine aspanate proteases cascade
  • the protective effects of Bcl-2 against apoptosis have been suggested to involve an antioxidant mechanism (29). although this is disputed based on the ability of Bcl-2 to block apoptosis caused by agents that are thought not to act by an oxidant mechanism (30) or caused by hypoxia (31 ).
  • WEHI7.2 cells transfected wiih trx formed tumors in scid mice that grew considerably faster than tumors formed by the wild-type parental celK or b nr/-2-transfected cells. This may be due. in pan. to a decreased rate of spontaneous apoptosis that occurred in ihe trx- transfected cell tumors.
  • High levels of Bcl-2 have been found in a wide variety of human cancers (34). Although transfection with bcl-2 is known to confer resistance to apoptosis induced by anticancer drugs and radiation, the effects of bcl-2 on tumor growth are less clear. Transfection with bcl-2 gives a survival advantage to cells in culture ( 351.
  • trx a gene found to be overexpressed in a number of human cancers, can inhibit apoptosis of cancer cells in culture induced by a variety of agents.
  • the rrx-transfected cancer cells show an increased growth, decreased spontaneous apoptosis, and decreased sensitivity to apoptosis induced by dexamethasone. If similar effects occur in patient tumors, then trx could be a new human proto-oncogene.
  • FIG. 1 Stimulation of human bone marrow colony formation by Cys73 ⁇ Ser mutant thioredoxin. Human bone marrow obtained as excess material from normal allogenetc bone marrow donors. The effects of Cys73 ⁇ Ser thioredoxin on colony formation by: (o) multilineage progenitors (CFU-GEMM); (•) erythroid progenitors (BFU-E); and (V) myeloid progenitors (CFU-GM), was measured over 10 days as described. Values are the mean of 4 dete ⁇ inations and bars are S.D.
  • CFU-GEMM multilineage progenitors
  • BFU-E erythroid progenitors
  • V myeloid progenitors
  • FIG. 1 Potentiation of IL-2 induced MCF-7 breast cancer cell growth by Cys73 ⁇ Ser mutant thioredoxin.
  • Cells were growth arrested for 48 hr in medium with 0.5% serum (10 s cells) then stimulated in the absence of medium with either IL-2 or Cys32 ⁇ Ser mutant thioredoxin at the concentrations shown. Cell number was measured after 48 hr. Each point is the mean of 3 deteiminations and bars are S.E. The dotted line shows stimulation by 10% serum.
  • FIG. 3 Inhibition of thioredoxin stimulated MCF-7 cell growth by receptor antibodies. Cell proliferation was measured as described in Figure 2. The concentrations of agents used were Cys73 ⁇ Ser mutant thioredoxin (Trx) 1 ⁇ M; monoclonal antibodies to FGF receptor, IL-2-receptor and EGF-recep * .or 4 ⁇ g/rnl; and EGF 10 nM. The EGF and EGFR were added as a negative control. Values are the mean of 3 detexi-iinations and bars are S.E. The dotted line shows the effect of 10% serum alone.
  • Trx Cys73 ⁇ Ser mutant thioredoxin
  • FIG. 2 Effects of Trx and C32S/C35S cDNA transfection on proliferation of MCF-7 cells.
  • Left panel 3 x 10* cells were plated in 3.8 cm 2 plastic culture dishes in DMEM with 10% fbs and cell number measured 7 days later.
  • Right panel 10* cells were plated in 2 cm 2 wells containing soft agarose and colonies measuring >60 ⁇ . counted 7 days later.
  • Control the Neol vector-alone tranfected MCF-7 cells; Trx 9, Trx 12, and Trx 20, MCF-7 cells transfected with human Trx cDNA; Serb 4, Serb 15 and Serb 19, MCF-7 cells transfected with C32/C35S cDNA.
  • Values are the mean of 3 determinations and bars are S.E. **indicates p ⁇ 0.01 compared to vector-alone transfected cells, •" ⁇ indicates p ⁇ 0.01 compared to vector-alone transfected cells.
  • Human thioredoxin is a putative oncogene that may confer both a growth and survival advantage to tumor cells. Over-expressed thioredoxin mRNA has been found in both primary human lung and colorectal cancers. To determine the intratumor distribution and amount of thioredoxin protein in human primary tumors and to determine if its overexpression is related to proliferation or apoptosis, we studied primary human gastric carcinoma samples. An immunohistochemical assay for thioredoxin in paraffin embedded blocks was developed. We studied ten patients with primary high risk gastric carcinoma.
  • thioredoxin protein overexpression to apoptosis we utilized a paraffin based in situ assay (Tunel) and to delineate proliferation we utilized the nuclear proliferation antigen detected by Ki67.
  • Thioredoxins are low molecular weight redox proteins found in both prokaryotic and eukaryotic cells (1).
  • the cysteine (Cys) residues at the conserved -Cys-Gly-Pro-Cys-Lys active site of thioredoxin undergo reversible oxidation-reduction catalyzed by the NADPH-dependent selenium containing flavoprotein thioredoxin reductase (2).
  • Human thioredoxin is an 11.5 kDa protein, with 27% amino acid identity to E. coli thioredoxin. It contains 3 additional Cys residues not found in bacterial thioredoxin that give it unique biological properties (3).
  • Thioredoxin was first studied for its ability to act as reducing cofactor for ribonucleotide reductase, the first unique step in DNA synthesis (4). More recently thioredoxin has been shown to exert redox control over a number of transcription factors, including NF-KB (5), FIHC (6), BZLF1 (7), the glucocorticoid receptor (8) and, indirectly through another redox factor Ref-1, AP-1 (Fos/Jun heterodimer) (7). Thioredoxin modulates the binding of the transcription factors to DNA and thus, regulates gene transcription.
  • Thioredoxin is also a growth factor with a unique mechanism of action.
  • the predicted amino acid sequence of thioredoxin is identical to that of a previously identified growth factor secreted by HTLV-1 transformed leukemic cell lines, called adult T-cell leukemia-derived factor (ADF) (3).
  • ADF stimulates growth of lymphoid cells (9,10).
  • human recombinant thioredoxin stimulates the proliferation of normal f ⁇ broblasts and human solid tumor cancer cells even in the absence of serum (1 1, 12). It does this by increasing the sensitivity of the cells to growth factors secreted by the cells themselves (13).
  • thioredoxin at nM concentrations increases the sensitivity of human breast cancer cells to interleukin-2 (IL-2) and basic fibroblast growth factor (bFGF) by 1000 and 100 fold, respectively (unpublished observations).
  • IL-2 interleukin-2
  • bFGF basic fibroblast growth factor
  • the term "voitocrine”, from the Greek “to help”, has been coined to describe this growth stimulating activity of thioredoxin (13).
  • Mutant redox-inactive forms of thioredoxin lacking the active site cysteine residues and E. coli thioredoxin are devoid of growth stimulating activity (12).
  • Human thioredoxin is known to be secreted from cells by a leaderless secretory pathway (15) so that it could be acting extracellularly to stimulate cancer cell growth.
  • thioredoxin is important for the growth, death and transformed phenotype of some human cancers. Stable transfection of normal fibroblasts with human thioredoxin cDNA (trx) increases their growth rate and transfection of human MCF-7 breast cancer cells with trx increases their colony formation in soft agarose (16). Transfection of the MCF-7 cells with a dominant negative redox inactive mutant trx causes inhibition of colony formation and almost complete inhibition of tumor formation when the cells were inoculated into scid mice.
  • Immunohistochemistrv Five micron thick sections were deparaf inized and then subjected to antigen unmasking with one of two methods with heat plus citrate buffer at pH 6.6 or microwave plus EDTA buffer at pH 8.0 as previously described (20,21). The best signal to noise ratio was established by judging reactivity with cell lines known to be a high expressor of thioredoxin (A549 human lung cancer) and a low expressor of thioredoxin (SK BR3 human breast cancer)(19).
  • the degree of thioredoxin expression in tumor cells was judged at 400x magnification as 4+ (very intensely positive), 3+ (moderately intensely positive), 2+ (moderate), 1+ (faint), or 0 (completely negative) throughout the sample.
  • a single investigator (TG) was responsible for scoring all the samples.
  • Ki67 proliferation antigens
  • biotin-avidin labelled method after avidin blocking 212.
  • the degree of Ki67 staining was classified as the percentage of nuclear positive tumor cells listed as: absent (0), >0-5% (+), 6-25% (++), 26-50 (+++), >51% (++++).
  • Apoptosis Assay Apoptotic cells were detected utilizing the TUNEL assay (23, 24) adapted to an automated in situ hybridization instrument (gen II, Ventana Medical Systems, Inc.).
  • the TUNEL assay utilizes recombinant terminal deoxynucleotidyl transferase (Tdt) (GLBCO BRL) for adding homopolymer tails to the 3' ends of DNA which are more abundant in apoptotic cells(23, 24).
  • Biotin-16, 2'-deoxyuridine-5'-triphosphate (Biotin 16-dUTP) was the label used for terminal transferase in this DNA 3' -end labelling reaction.
  • Avidin-Horseradish Peroxidase and 3,3'-diaminobenzidine as chromogen(23, 24).
  • the instrument utilized deparaffinized sections with subsequent digestion with Protease I (Ventana Medical Systems Arlington, Az.) for 8 minutes VMS1). Incubations were performed per Ventana Gen II protocol on the instrument with the final steps being as above using avidin-horse radish peroxidase and DAB detection method to visualize the apoptotic nuclei as an intense brown color (diaminobenzidine). As an enzyme control we utilized two sections from each tissue: one with Tdt
  • the TUNEL assay result was scored by the number of brown - apoptotic tumor nuclei per high power field (400x objective). The values were: 0 (absence of apoptotic cells), + (>0-2/hpf), ++ (2-4/hpf), +++ (>4-8/hpf), ++++ (>8/hpf).
  • Thioredoxin expression was correlated with Ki67 expression and with apoptosis measured by the TUNEL assay using Spearman's nonparametric rank correlation test.
  • the optimum signal to noise ratio was found by using the following antigen retrieval conditions: microwaving at pH 8.0 in EDTA as tested by a high thioredoxin expressor (A 549) and low thioredoxin expressing (Sk BR3) cell line.
  • the lower level cells in the pits showed cytoplasmic and scattered nuclear staining, while the higher mid-level graduation staining was typically lighter and restricted to the cytoplasm. The significance of this differential distributions is not known.
  • Thioredoxin does not have a known nuclear localization sequence (3).
  • thioredoxin is specifically located within neoplastic gastric carcinoma cells and not in stromal cells or admixed B or T lymphocytes or macrophages.
  • the tumor cell thioredoxin density typically exceeded that of the adjacent normal mucosa.
  • the hematoxylin and eosin stains reveal a pleomorphic carcinoma invading the gastric wall.
  • the thioredoxin expression (right upper and lower panels) is present in both the nuclei and cytoplasm of tumor cells in malignant glands and in rare associated leucocytes. Thioredoxin expression is absent in the adjacent stroma (lOOx to 400x).

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Abstract

L'invention concerne l'utilisation de thiorédoxine, entre autres en tant que stimulateur de la croissance cellulaire, de même que pour cribler des agents utiles dans la réduction ou la prévention de l'inhibition de l'apoptose associée à la thiorédoxine, ainsi que des agents utiles dans l'inhibition de la croissance cellulaire stimulée par la thiorédoxine.
EP97952292A 1996-12-06 1997-12-05 Utilisations de thioredoxine Withdrawn EP0946192A1 (fr)

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US3199596P 1996-12-06 1996-12-06
US31995P 1996-12-06
PCT/US1997/022292 WO1998024472A1 (fr) 1996-12-06 1997-12-05 Utilisations de thioredoxine

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US6552060B1 (en) 1997-08-11 2003-04-22 Prolx Pharmaceuticals, Inc. Asymmetric disulfides and methods of using same
US6372772B1 (en) 1997-08-01 2002-04-16 Prolx Pharmaceuticals Corporation Inhibitors of redox signaling and methods of using same
IL137177A0 (en) 1998-01-30 2001-07-24 Genesense Technologies Inc Oligonucleotide sequences complementary to thioredoxin or thioredoxin reductase genes and methods of using same to modulate cell growth
AU2003263795A1 (en) * 2002-08-02 2004-02-23 University Of Rochester Thioredoxin mutants and uses thereof

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SE8903003D0 (sv) * 1989-09-12 1989-09-12 Astra Ab Novel medical use

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