WO1998024472A9 - Utilisations de thioredoxine - Google Patents

Utilisations de thioredoxine

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Publication number
WO1998024472A9
WO1998024472A9 PCT/US1997/022292 US9722292W WO9824472A9 WO 1998024472 A9 WO1998024472 A9 WO 1998024472A9 US 9722292 W US9722292 W US 9722292W WO 9824472 A9 WO9824472 A9 WO 9824472A9
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WIPO (PCT)
Prior art keywords
thioredoxin
cells
trx
human
cell
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PCT/US1997/022292
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English (en)
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WO1998024472A1 (fr
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Priority to EP97952292A priority Critical patent/EP0946192A1/fr
Priority to CA002274116A priority patent/CA2274116C/fr
Publication of WO1998024472A1 publication Critical patent/WO1998024472A1/fr
Publication of WO1998024472A9 publication Critical patent/WO1998024472A9/fr
Priority to US12/694,577 priority patent/US20100166724A1/en

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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, inter alia, a cell
  • tumor cell growth in a tumor cell that over-expresses thioredoxin comprising contacting said
  • Such agents can include, inter alia, small molecular compounds that complex
  • Such agents can include, mter 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
  • NTH 3T3 cells In a non-limiting embodiment of the present invention, NTH 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 Ref- 1. activator protein- 1 (Fos Jun heterodimer. Ref. 4). Cloning and sequencing of human Trx have shown that it has a predicted ammo acid sequence (5. 6 identical to that of a growth factor secreted by virus-transformed leu emic 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 adde Trx is not taken up by cells ( 1 1 ) and appears to stimulate cell growtr
  • Trx by enhancing the action of other growth factors ( 1 1. 12).
  • the activity of Trx is required for growth stimulation, and mutant Trxs do not stimulate cell growth ( 10).
  • Trx mRNA levels are increased compared with corresponding normal tissue in almost half human p ⁇ mary 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 unequivocally demonstrated that endogenousl 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 ( Rockville. MD). maintained in DMEM containing I0 ⁇ * FBS under 6 r c CO : at 37*C. and passaged before confluence. NIH 3T3 cells were transfected with Trx:pRXneo. Trx. pDC304neo. C32S/C35S.pDC304neo. or pRXneo alone. MCF-7 cells were transfected with Trx:pDC304neo. C32S/C35S:pDC304neo. or pDC304neo alone.
  • Transfection used liposomes of .V-( l.2.3-dioleolyi)propyl
  • Cells were selected by growing for 4 weeks in DMEM with 10*1 FBS and 400 ⁇ g/mi G4I8 sulfate (Life Technologies. Gaithersburg. MD). Cell colonies were isolated by tr psimzati ⁇ n onto small squares of stenle filter paper and expanded by growing in the s me medium. All studies were conducted on clonal ceil lines between passages 3 and 10.
  • Fig. Z Effects of tranifectioo with T ⁇ or C32S/C35S cDNA on the growth of NIH 3T3 cells. Ceils were plated in plastic dishes at a density of I x 10* cells/c ⁇ r in DMEM with lOt FBS and cell number measured daily. •. NeoC vector alone-transfected cells. The apparent decrease in the number of cells after day 3 et due to detachment of cells from the plastic surface: V. ⁇ . and i ⁇ . Thrao. TtuoAD. and Tluo9 cells transfected with Tn cONA: D and ⁇ . NIHBH and NTHBF cells transfected wnb C32S J5S cONA.
  • Tumor formation b ⁇ transtected NIH 3T3 cells was studied b> the s.c injection of 10' transtected cells in ⁇ . l ml ⁇ f ste ⁇ le 0 ur NuCI into ihe bucks of groups ⁇ f tour male SCID mice or six nude mice.
  • Tumor formation by MCF-7 cells was studied b> injecting : ⁇ 10' ceils in 0.1 ml of sterile 0.9 ⁇ " r NaCI and 0.1 ml of Matnsel i ⁇ e ton Dickinson. Bedford. MAi s.c. into the backs of groups of four temale SCID mice that had been implanted s.c. 2 da s previously with 21 -day release pellets of 0.2.
  • Trx and C32S C35S Transfection of NIH 3T3 Cells Transfection of mouse NIH 3T3 cells with TrcpRXneo yielded 6 clones stably expressing Trx mRNA. transfection with T ⁇ :pDC304neo yielded clones, and transfection with C32S/C35S:pDC304neo yielded 12 clones. The levels of transfected mRNA in some of the clones is shown in Fig. 1. The human Trx and C32S C35S mRNAs were larger than the endogenous mouse Trx mRNA.
  • transfected Trx mRNAs probably because the transfected Trx mRNAs also contain portions of the vector promoter region, the 3' leader sequence, or the polyadenylaie tail.
  • the level of transfected Trx mRNA expression was relatively low, being only 0.2- 1. -fold the endogenous mouse T ⁇ mRNA.
  • Western blotting showed no significant increase in the level of T ⁇ protein in the cells compared with wild-type or venor alone-transfected cells (results not shown).
  • 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-transfected ceils Neither-the vector alone-transfected NIH 3T3 cells nor the Trx or C32S/C35S Trx transfected cells formed colonies in soft agarose (results not shown ) .
  • Trx-tran>.fected NIH 3T3 cells Thioo or ThioAD were injected i.m. into SCID or nude mice, there was no tumor formation over 40 days (results not shown).
  • 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 cells 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 in 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 mRNA up to 2 1 -told the endogenous T ⁇ mRNA levels. Light microscopy showed no difference in the appearance of vector alone-transfected and Trx- transfected MCF-7 cells growing on glass coverslips ( Fig 5 ). and both were similar to wild-type MCF-7 cells. In contrast. C32S/C35S- transtected MCF-7 cells appeared more rounded and had a 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 alone-transfected MCF-7 cells (Fig. 6). However, when grown in the absence of. or with 0.5 ⁇ FBS for 2 days, the Trx-transfected cells grew at twice the rate of the vector alone-transfected cells (results not shown).
  • the C32S/C35S-trans- fected cells grew at a significantly slower rate that was 56-78 c ⁇ of the 24472
  • Trx- and C3-S/C35S-transfected MCF-7 CtUs Tumor Formation by Trx- and C3-S/C35S-transfected MCF-7 CtUs.
  • the vector alone-transfected MCF-7 cells injected into SCID mice formed tumors that grew at the same rate as nomransfected MCF-7 cells we have seen in other studies.
  • Trx-transfected MCF-7 cells formed tumors in SCID mice, although they grew at a significantly slower rate than tumors formed by vector alone-transfected •ells- 57% for Trx 12 cells and 381 for Trx 20 cells (both P ⁇ 005 by least squares regression analysis. Fig 7).
  • Trx regulates the redox state and -c itv ot a number of intracellular proteins that control cell growth, including nbonucleotide reductase ( 11 and the DNA binding of several transcription factors (2-41 Recombinant human Trx added to normal and cancer cells in culture stimulates their proliferation ( 111. However, it has not been demonstrated that endogenously produced Trx can stimulate cell proliferation. Furthermore, the role Trx may play in malignant transformation of cells is not known The present study was undertaken to address some of these questions NIH 3T3 cells transfected with Trx 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 tumors when inoculated into immunod- crucie.
  • the Trx-transfected MCF-7 ceils did not show in ⁇
  • Trx secreted by the transfected MCF-7 cells might promote some immune rejection, even by the SCID mice, which, although deficient in mature B and T lymphocytes, have natural killer-, myeloid-. and antigen-presenting cells ( 26 ⁇ Both NIH 3T3 and MCF-7 breast cancer cells transfected with the C32S/C25S Tra showed slowed growth rates on a plastic surface In addition, colony formation by MCF-7 breast cancer cells in soft agarose was considerably decreased. When injected into SCID mice , the C32S/C3JS-transfected MCF-7 cells formed only microscopic tumors.
  • C32S/C35S is a redox-inac e mutant Trx that acts as a competitive inhibitor of Trt reductase ( 10).
  • Trt reductase 10
  • Our X-ray crvstallo- graphic studies have identified a highly conser ed 12 ammo acid hydrophobic surface on mammalian, but not bacterial. Trxs. which
  • MCF 7 breast cancer cells SCID mice Fema SCID mice implanted s c - davs rarsnuslv with a l-uav release pellet ot 0 23 m
  • Trx transiected tells t Tn .O
  • II-tniufccied cells ⁇ Serb 4 CJIS OSS uansfected cells
  • Serb 15 CJ-S CISS-transfected cells There were four mtce pet group Tumor growth was measured twice a week tor 40 davs The 17 0-es ⁇ rad ⁇ ol pellet was replaced at 21 davs Values are mean Ban SE
  • 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-active substrates of Trx Whatever the mechanism, it appears that C32S/C35S acts in a dominant-negative manner to inhibit the effects of endogenous Trx and. tn so doing, inhibits cell growth and reverses the transformed phenotype of MCF-7 breast cancer cells
  • Trx or C32S C35S protein that is produced by the transfected cells appears to be secreted into the medium Whether the transfected Trx is produced in a different compartment to endogenous Trx. allowing it to be secreted, or whether a constant proportion of Trt is secreted is not known Tn is known to be secreted from cells by a leaderless secretory pathwav ( 17).
  • the concentrations of Trx found in the medium up to 10 nvi after 6 h.
  • Trt at nanomolar concentrations will potentiate the growth effects of cytokines such as ⁇ n ⁇ erieuk ⁇ 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 It should be noted that Trx binds to the surface of cells ( 1 1. 28) so that secreted Trx could have a local effect at the outer cell surface although concentrations in the medium are low
  • Trx mRNA levels in cells were not high, only up to I -fold endogenous Trx mRNA levels, and were independent of the mammalian transfection vector used Typicallv mRNA levels resulting from transfection using such vectors are 10-50-fold or higher (29) It mav be that high levels of Trx gene expression are toxic to cells We have found onlv a low expression of the human Trt gene tn PC17US97/22292
  • transgenic mice '1 In ⁇ ntraM. some human tumors show very hiyh levels ot Trx. RN ⁇ .ompared vwih the normal tissue' mure than 1 l -told in humjn p ⁇ mar. lung tumors 130) and even higher in human pnmarv colon tumors ⁇ 1 » It is not known why higher Trx RN ⁇ levels could not be obtained in transtected cells, v e ⁇ ere unable to obtain transformation ot NIH 3T3 cells with Trx. It remains to be demonstrated whether the much higher levels ot Trx expression seen in some human tumors might be transforming.
  • ⁇ l a ise te cyMcin in human thmminxi ⁇ r*v*k*.ev inhih tt ors o l human i hio ⁇ 'Unxin reUuctj.se and elimination nl rnmrccriic prnpe ⁇ ies ⁇ l t hioredimn •> J Bnil Ch m . .'ov 1 1714-1 1720. Is»*4
  • 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 (Trx ) is a widelv di s tributed redox protein chat reyulaces *everai mc ⁇ i elluiar redox - •ce.se> and stimulates che proliferation of both normal and tumor cells. W hjve found mat when ibser.ce of reducing airents. human recombinant Trx undergoes spontaneous oxidation, iosinv.
  • Trx forms a dimer that is stabilized by an intermole ular C'.s *-Cvs ' .
  • a Cvs 7 ' ⁇ Ser mutant Trx (CT3S ) was prepared to determine the role oi Cys 71 in oxidative rowth stimulation.
  • C73S was as effective a» Trx in s mulattne cell growth and was a comparable ⁇ uoredoxin reductase.
  • C73S did not show spontaneous or oxidant-induced loss of acnvicv and did rter.
  • Trx can exist in monomenc forms, some of which are mediated bv not stimulate cell proliferation but can be reduced by thioredoxin reductase. Cys 71 is also involved o( an enrymatically inactive homodimer. which occurs on long term storage or by chemical .us. although clearly involved in protein inactivation. Cys' 1 is not necessarv for the growth tivitv of Trx.
  • Trx is a redox protein found in both eu aryotes and p ⁇ karyotes [I].
  • 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 by thioredoxin reductase (2J. Trx was originally identified in EJ C /V ertcru cab as a hydrogen donor for rib ⁇ nucleotide reductase PJ.
  • Trx has since been found to act as an intracellular dithiol-disulfide reductase and to modulate the activity of a number of intracellular proteins (4-6] including the DNA binding of transcription factors (7-10). Trx-like sequences are found in other proteins including protein disulfide isom- erase (11). There is evidence that Trx may play a role in che growth and transformed phenotype i some cancers. Trx is
  • Trx is secreted from cells bv a leaderless secre- torv pathway [19] and could he acting as an autocnne ractor tor the growth ot some cancer cells [IS)
  • Trx unlike human Trx. does not stimulate the growth or human solid cancer cells [ 18)
  • the structures of E c ⁇ u. ind human Trx are similar, and Kith .ire substrates tor human thioredoxin reductase.
  • the surface residues of the two forms varv considerablv [20
  • Human Trx has 3 additional cysteine residues.
  • Cvs 62 , Cys 0 " and Cys in addition to those in the active site, which do nut normally form intramolecular disulfide bonds [20, 21).
  • Trx can also form a homodimer with a 1100 A : inter ace domain and a disulfide bond between Cys 7 ' from each monomer [20].
  • Trx During our studies of cell growth stimulation by Trx we observed that storage of the Trx without a reducing agent for even a few days resulted in a loss of its growth-stimulating activity, although the Trx ' remained a substrate for reduction by thioredoxin reductase. We have, therefore, examined the role of spontaneous and induced oxidation of Trx and cysteme-deleted mutant Trxs, and their ability to stimulate cell proliferation.
  • Cvs' ⁇ Ser mutant human Trx (C73S) was prepared from single-stranded, sense strand human Trx cDNA hgated by polyethylene glycol pre ⁇ ptta- non into the Bluesc ⁇ pt KS vector (Stracagene. La Jolla. CA) using R408 helper phage.
  • the single-stranded cDNA was used for oligonucleotide-directed m vitro mutagenestj (Version 2.1 Kit, Amersham. Buckinghamshire.
  • Trx bv dtdeox ⁇ sequencing C73S Trx wits expressed and punfied as previously described [16). All Trxs were stored at -20° JS a 25 ⁇ I stock solution in 5 mM DTT. Before use. the DTT was removed bv passinc the Trx solution through a PD-IC desalting column (Pharmacta, Uppsala. Sweden). The Trx solution was kept at 4° and used within 1 hr (fresh) or stored m water or 0.1 M potassium phosphate-buttered 09% N CI at 4" or -20* tor specified times.
  • Ox ⁇ d ⁇ :ed Trx for cell growth studies was prepared by adding a 5-told molar excess of H 0 : to a 25 ⁇ M Trx stock solution without DTT and 1 hr later removing unreacted H G ; using a PC *1 - 10 column.
  • Trx ana modified Trxs >n MCF 7 cell growth was determined as previousK de.cr ⁇ ed [IS] Brietlv, 10' cells were plated in a 35-mm culture dish .n DMEM containing 10% fetal bov ine serum and. arter attachment tor 24 hr, growth arrested using DMEM with 0 5% fetal bovine serum for 48 hr The medium was tnen replaced with DMEM containing Trx or other additions for 2 davs and cell number measured with a hem ⁇ cvtometer. All incubations were conducted in tnplicate
  • Trx reduced/min/mg protein Human placenta thioredoxin reductase. specific activity 33.3 ⁇ mol Trx reduced/min/mg protein, was prepared a previously described [231 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 descnbed by Luthman and Holmgren [2].
  • Trxs that had been aged at room temperacure for 48 hr, 7 davs.90 days, or Trxs treated for 1 hr with 1 mM diamide, 10 mM DTT, 3 mM 2-mercaptoethanol or 2.1
  • Cys ' -* Ser mutant Trx stimulated the proliferation of human MCF-7 breast cancer ceils
  • the EC W ° r growth stimulation C73S was 350 nM and the maximum effect was seen it 1 ⁇ M which is similar to values we have previousK reported tor stimulation ot MCF-7 cell proliferation bv recombinant human Trx [IS
  • FIG. 1 S ⁇ nula ⁇ on of MCF-7 breast cancer ceil f-rowth by fresh and aged Trx and C73S.
  • MCF-7 ceOs were powth arrested and the stimulation of celt profile-ration matured over 2 davs using 1 uM Trx or C73S that was fresh or lied brsn stored as a 23 ⁇ M stock solution without leducmt agent for 3 davs or 90 days at 4°.
  • Also shown for resetence is the effect of 10% fetal bovine serum. Each value is the mean of 3 dete ⁇ ninadons, and bars are SEM.
  • Trx stored 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 pbc ⁇ v ral thioredoxin reductase with a K. of 0.20 ⁇ M and a V mm of 6.3 tun ⁇ l/rain/ ⁇ g. These values are similar to those we have pisrviously found for fresh Trx, which were a i ⁇ , of 0.33 ⁇ M and a ⁇ of 5.9 rund/min/ ⁇ g (231-
  • Trx without DTT on is ability to act as a substrate for rhioredoxin reductase was hnvesogated (Table I).
  • Trx When stored in H : 0 either at -20* or at room ternperature Trx showed a loss of activity with a hatf- fe o
  • Trx showed a slow, delaved reduction bv thioredoxin reductase that was stimulated by catalv ⁇ c amounts ot rresh Trx (Fin. 1). It is important to note that the loss of activity of Trx as a substrate for thioredoxin reductase was much slower than the loss of activity as. a stimulator ⁇ i cell growth. C73S did not show a loss of activity as substrate for thioredoxin reductase upon storage for up to 3C davs.
  • Trx The ability of Trx to act as a substrate for thioredoxin reductase was completely inhibited bv treatment wirh 5 molar equivalents of H.O;, whereas C375 remained ullv active after treatment with 100 molar equivalent; ot H.Os (Fig. 3).
  • Electrophoretic analysis of freshly prepared human Trx stored in DTT showed a mixture of 5 bands of apparent molecular weights ranging from 8.1 to 11 kDa (Figs. 4, 5. and 6, lane 1 ).
  • Storage of Trx ar 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.
  • apparendy, to a Trx dimer (Fig. 4, lane 3).
  • Storage of Trx without DTT for 90 days at 4* resulted in almost complete conversion to the Trx dimer (Fig. 4. lane 4).
  • Trc Reduction of aged Trx bv thioredoxin reductase.
  • the incubation mixture contained 0.1 M HEPES buffer. pH 7.6. 5 mM EDTA. 17 ⁇ M insulin. 100 ⁇ M NADPH. 15 PR/ml human thioredoxin reducnu*. Tr ces »ho the on- dabon of NADPH at 340 nm at room temperature i Ui « A. 1 uM froh Trx: B. 30 nM fresh Trx: C. W ⁇ Y « «ed 1 uM T ⁇ c D. 1 ⁇ M 90-day aged Trx and 30 nM rresh int.
  • FIG. 3 The effect of H 2 0 2 on the reduction of Trx (filled bars) and C73S (open bars) by thioredoxin reductase.
  • Trx solutions were treated with varying amounts of H 2 0 2 for 18 hrs at room temperature.
  • Reductase activity was measured by adding treated samples to a solution of 0.1 M HEPES buffer, pH 7.6, 5 mM EDTA, 17 uM insulin, 100 ⁇ M NADPH, 15 ⁇ g/ml human thioredoxin reductase and measuring the rate of NADPH oxidation at 340 nm at room temperature.
  • Trx dimer following diamide treatment was also confirmed by gel permeation chromatography (results not shown).
  • HJCN treatment of Trx also caused dimer- iati ⁇ n but produced a different banding pattern to that produced by diamide (Fig. S. lane 6).
  • the studv shows that human recombinant Trx undergoes a t least 2 levels of spontaneous and induced oxidative trans ⁇ formation.
  • the first oxidation occurs spontaneously with i n a few days to a form(s) that can no longer stimulate ceil growth but remains a substrate for thioredoxin reductase.
  • the slower oxidation occurs over a pe ⁇ od of weeks, or can be induced bv 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 bv catalase and are reversed bv the thiol reducing agent DTT us consistent with the interpretation that the changes n 'Trx are due to oxidation.
  • Cys 7 ' appears to plav a c ⁇ ncal role in both levels of oxidant- induced mactivanon 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 a 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. Pro- tein was stained with silver stain. Lane 1, fresh Trx: lane 2, Trx 48 hra at room temperature without DTT; lane 3. Trx 7 davs at room temperature without DTT; and lane 4, Trx stored 90 davs at 4* without DTT. Position of molecular man markers in kDa are shown on the left. 98/24472
  • the number of free chi ⁇ ls in fre>h Trx was determined to be 4.5 to 4.6/molecule by EI1- man ' .s reauent [2S] (data not shown), indicating chat all five cvstemes are in the sulfhydrvl form.
  • Treatment of NEM- alkvlaced Trx with oxidizing or reducing agents produces no change tn rhe banding pattern (data not shown), which is further evidence that all 5 sulfhvdrvls have been alkvlaced.
  • FIG. 6 Oxidation and reduction of mutant T ⁇ studied bv SDS-PAGE.
  • Lane 1 fresh Trx- lane 2.
  • Position of tnoiecular mass markers m kDa are shown on the left and right.
  • Trx may he undergoing "native" intramolecular disulfide bond formation pnor to elextr ⁇ phoresis, which preveno the formation of random disutf k'bond formation seen with denaturation and ctectntphuiesis of fresh Trx.
  • bovine pancreanc trypsin inhibitor [27, 31].
  • FIG. 7 Position of cysteines in human Trx. Ribbons and ball-and-s ⁇ c representation showing the relative positions of Cys J2 , Cys". Cys". Cys 1 * and Cys' 3 , based on the crystai coordinates for the wild type reduced protein (20). None of the tniols are in a position for rmaitnoV bond t rma ⁇ on except for the redox active pair Cys 32 and Cys 3 *. The inter- moiccuiar dtwilfide bond ranming the les * distortion in the protein would be between Cys 32 and Cys 71 .
  • Trx homodimer If a solution ⁇ f Trx is left long enough, ⁇ r upon treatment with a strong oxidizing agent such as diamide or H,0,. there is formation of a 23-lcOa Trx homodimer. Reduction of the Trx dimer by thioredoxin reductase is slow and de- laved, and is stimulated by low c ⁇ ncentran ⁇ ns ⁇ f fresh Trx. suggesting there may be an autocatalvtic process. A similar conclusion was reached by Ren et al. (34). Formation of the Trx homodimer appears to involve the Cvs' 1 residue since C73S. where Cys' ' is replaced with se ⁇ ne.
  • Trx un erBoes a faster loss of ac ⁇ virv with thioredoxin reductase in PBS versus, water indicates that iron-induced oxidation or jn increase in ionic strength mav stabilize and enhance dimer formation, which is consistent with the hvdrophnbic nature of the dimer interface observed in crvstals of human Trx.
  • the importance of "the monomeric oxidativ e rormis) or Trx is, unknown. While the structural nature is vet to be identified, it does have different biological ac ⁇ vitv in our m vitro system. Trx is secreted bv cells into the extracellular environment, which is predominantiv oxidizing, and might be expected to undergo monomenc oxidation.
  • Trx homodimer has been repo ⁇ ed in diamide-treated Jurkat cells [35].
  • Trx homodimer has been repo ⁇ ed in diamide-treated Jurkat cells [35].
  • Trx is believed to exist in normal cells at concentrations from 1 co 10 ⁇ M [2, 12], chough in selected tissues and specific cell compartments this value could be much higher. It is therefore not unreasonable to assume chat Trx will undergo homodimer formation in vivo. As we observed with the enhanced inactivation of Trx in phosphate buffered saline, we expect dimer formation to precede faster m vwo than we observe m vitro in water. Whether dimer formation m two would prevent the faster oxidation to an intramolecular fotm is unknown. The slow aut ⁇ catalvtic reducnon of che 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 oxidant-induced conversion to a form(s) that doe not stimulate cell proliferation, but is still a substrate for reduction bv thioredoxin reduccase. There is much slower (over ⁇ penod of weeks) spontaneous oxidacion of Trx to a Cvs ⁇ , -sta lized homodimer form that is not a substrate for thioredoxin reductase and chat also does not stimulate cell proliferation. Both conversions can be reversed by treatmen t with the thiol reducing agent DTT. and both appear to involve the Cys 7 ' residue. A Cvs v " ⁇ Ser mutant Trx.
  • Trx which st imulates cell pn>liferation and is JS effective a substra t e for t hioredoxin reductase as Trx. did not show a g e ot oxida t ion-induced loss of these activities.
  • Trx uraduallv loses i t s abilitv to stimulate cell proliferation and t o be a subs t ra t e for thioredoxin reductase. unlike the C ⁇ s " ; ⁇ Ser mu t ant Trx. which retain- these activities wirh no l s
  • Cvs 7" ' is not critical tor hu-U.-ic.il ac 8/24472
  • the redox protein thioredoxin plays an important role in controlling 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 WEHI7- ⁇ thymoma cells.
  • WEHI7.2 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 thapsigargin, but not by A'-acetyl-spbingosine.
  • the trx-transfected cells formed tumors that showed increased growth compared to wild-type, as well as icf-2-t ⁇ ansfected, WEHJ7.2 cells.
  • the trx- and ⁇ c/-2-transfected cell tumors both showed less spontaneous apoptosis than tumors formed by the wild-type cells.
  • m-t ⁇ insfected 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- karyoiic and eukaryouc cells (1).
  • the cysteine residues at the conserved Cys 32 -Gly-Pro-Cys 35 -Lys active site of Trx undergo reversible oxidation-reduction catalyzed by the NADPH-dependent selenium- containing flavoprotein Trx reductase (2).
  • Human Trx is a protein of M ⁇ 11.500 with 27% amino acid identity to Escherichia coli but containing three additional Cys residues not found in bacterial Trx that give the human proiein 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 exert redox control over a number of transcription factors, including nuclear factor KB (5), transcription factor IHC (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 ftbroblasts and a wide variety of human solid and leukemic cancer cell lines (10. 11).
  • Redox activity is essential for growth stimula ⁇ on by Trx.
  • mutant redox-inactive forms of Trx lacking the active site Cys 32 and Cys 35 residues are devoid of growth stimulating activity (11).
  • Studies with '" -labeled Trx have revealed no high-affinity binding sites that
  • Trx Trx.
  • thioredoxin scid. severe combined immunodefi- cte ⁇ i CAT rhlnramnhenicnl acetvltransferase might suggest receptors for Trx on the surface of cancer cells (12). Trx appears to -vnw'f ceD prolifc ⁇ rion by increasing the sensitivity of the cells to growth factors secreted by the cells rtanselves (12).
  • Trx mRNA is elevated compared to paired normal tissue in almost half of the human primary lung and colon ⁇ unors we examined (3, 13).
  • Other studies have found increased Trx in human neoplasric cervical squamoua epithelium cells and hepato- cellular carcinoma (14, IS).
  • Trx overexpresskn may be a factor in the growth of some human cancers.
  • Trx gene expression is decreased during dexamesJvasone-induced apoptosis of mouse mym ⁇ rna-derived WEH17.2 cells (17).
  • Trx cDNA was stably transfected with human Trx cDNA and examined the effects on both spontaneous and drug- induced apoptosis in vitro and with the cells growing in scid mice.
  • Oil Human wild-type Tn cDNA was utcyed as described prevtrjuaty. ctoned into the NoA site of the pDC304oeo mammalian trat sfecoon vect ⁇ r ( 16) and transfected by etactroponron ii ⁇ ⁇ rju ⁇ WEW cells (18). Transfected cells were maintained as culture densinet up to 10* ceUs ml in DMEM containing 10% fetal bovtne serum surjokroeased with 800 ⁇ g/ml G418 sulfase. and clones were isolated in soft agarose and then maintained in 200 ⁇ g ml G418 sulfate.
  • Glwacnrttrntd Receptors The level of ftmrnonal ghtcorxnscoid receptors was assessed using a transient cotransfecoon of cells with a giucocorocoid response elensent CAT reporter plasmid (prnrnCAT; Ref. 20) and -galacso- ndaae. After a 22-h recovery period, the cells were treated with I ⁇ M dexa- rnexhasone. and CAT proton was measured after an additional 24 h using a CAT El, ISA (Boenringer Maratbeirn. Indiaiurpoiis. IN). An aliquot of the n-ansfecied cells was stained for 0-galac ⁇ os ⁇ da ⁇ acnviry and CAT activity normalized for transfection efficiency.
  • Apoptosis was measured by an EUSA for hmone-associated DNA ftagments (21). by ⁇ orphotogy and by flow cytometry (22).
  • the criteria used for the morphotogical tden ⁇ ficauon of apoptooc cells included condensation and rnarginaDo ⁇ of the chrotnann with the formation of crescents, cell shrinkage, increased staining, nuclear n-agrnentation. cytoplasreic vacuoliza- bon. and apoptooc body formation. Cells were incubated with 20 ⁇ g/ml 7-am ⁇ no acnnomycm D for 30 mm at 4*C before being analyzed by flow cyiornerry.
  • Tumor t ⁇ rmaiion bv wild-tvpe and transtected WEHI7 2 cells was studied bv injecting 2 x 10 7 cells in 0 I ml ot mat ⁇ gel s c into the flanks ol groups ot 20 tem ⁇ le scid mice Tumor volume was measured with calipers and mice were euthanized when the tumor volume exceeded 2 cm"
  • Nine rouge alter tumor cell injection 10 mice from each group were injected i p with 1 mg/kg/dav dexamethasone in lO'r ethanol in 0 9 NaCI Control mice were injected with vehicle alone On 14 three mice trom each group were euthanized with CO. and the tumors were excised and immediately fixed in gluiaraldehsde
  • Tissue for Bright-Field Examination The glutaralde hvde-fixed tissue was postttxed in osmium tetroxide dehvdrated in a graded se ⁇ es ot alcohols and embedded in epoxv resin One- ⁇ m-thick sections were prepared and stained with loluidine blue for b ⁇ ght-field examination
  • Trx-hke fluorescent staining was observed in the nucleus as well as the cytoplasm of the cells (Fig I d In the wild-type cells 60 1 r 5 1 % of the fluorescent staining was in the nucleus, in the Trx5 cells it was 59 8 - 2 5%. and in the Trx6 cells it was 36 1 r 1 8*
  • the trr-transfected WEHI7 2 cells were resistant to apoptosis induced bv 1 ⁇ M dexamethasone as measured by histone-associated DNA fragmentation (Fig 2A > or bv flow cytometry (Fig IB) Histologtcal examination of the WEHI7 2 cells revealed a classic apoptotic morphology in response to dexamethasone
  • Fig 2A > or bv flow cytometry Fig IB
  • Histologtcal examination of the WEHI7 2 cells revealed a classic apoptotic morphology in response to dexamethasone
  • 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 i We also studied the effect of trx trans- tection on
  • Fig 1 Northern blot htbnduaoon analvsts ol total RNA extracted from wild-type mouse W EHI” : cells Irom pDC.VUoeo »ect ⁇ r alone transfected WEHI7 2 cells (Neol. ind Irom the rrt-transfected W EHi * 2 clones Trx? and Tnb full-length " : P-labeled rrr cDN p ⁇ ce was use tor hstindj/aiion fp nam transiected human Tr* mRNA.
  • Trx mRN ⁇ The same-, on the neti how the position of molecular weight markers ⁇ kb ⁇ B fluorescence tmmun ⁇ rtistochemicai staining of Trx in cells using tmmu- noalfinirv-punficd rateHt anuhuman Trt polvclorui anubodv. bionnvlated goat antirabbit IgG fluorescetn ttnrputidin and laser scanning ontocal microscopy /. wild-type W EHt" : cell>. .'. pDCJOaneo >ec ⁇ or alone iransiecied WEHI7 2 cells. J.
  • TrrS trx- translecteu cells J Tr ⁇ f> rrr transiected cells c fluorescence immunohistochemical staining ol Tn using Cs .
  • Fig. 2 Effects of trx and bcl-2 transfection in WEHI7.2 cells on deiamethasone-induced apoptosis.
  • ⁇ apoptosis measured by an E 1SA for hinonc- associatied DNA fragments, expressed as relative nucleosomal enrichment.
  • the /rr-transfected WEHI7.2 cells formed tumors that grew more rapidly than tumors formed b> either wild-type or 6c/-2- ⁇ ransfected WEHI7.2 cells (Fig. 3 ⁇ ).
  • tumors formed by the wild-type cells showed fields of apoptouc cells adjacent to fields ot viable cells, as well as apoptotic cells admixed with viable-appea ⁇ ng cells (Fig 3 ⁇ )
  • the cells undergoing apoptosis exhibited the classic appearance of condensed and marginaied chromaiin. some in the form of crescents, and a dense cytoplasm accompanied by vacuolizaiion.
  • the /n-transfected WEHI7.2 cell tumors showed minimal numbers ot cells undergoing Table I Effect of tn and bcl-2 transfection on apoptosis induced bv different agents pDC304neo vector-atooe-transfected W ⁇ HT7.2 cells (Neo). *c/-2-transfected WEHT7-2 cells (W.Hbl2). and TrxJ and Trx6 r ⁇ r-traastected WEHI7.2 cells were treated with 1 UM dexamethasone tor 48 h. 100 ru. staurotpo ⁇ ne for 21 h. 100 ⁇ tt ⁇ J-aceryl-sphing ⁇ sine for 24 h. I UM etoposide for 15 h.
  • rut thapsigargin for 24 h.
  • the umes were determined to be optimum for detecting apoptosis with each agenL Apoptmu was measured by flow cytometry as in Fig. 2S.
  • Statistical analysis was by linear regression with indicator values for drugs and cells using the Stata sta ucal package IS tat Corp.. College Station. TXl
  • Tumors formed by _>c7-2-transfected WEHI7.2 cells also showed very few cells undergoing apoptosis (not shown). Areas of necrosis were seen in wild- type, rrx-transfected. and £>c7-2-transfected cell tumors, usually adjacent to fields of viable-appea ⁇ ng tumor cells or, in the case of the wild-type cells, adjacent to areas that show extensive apoptosis or next to viable-appea ⁇ ng cells.
  • Fig 3 A tumor formauon tn scid mice by wild-type WEHI7 2 cells (O and •). oc/-2-trans- fected WEHI7.2 (W Hbl2) cells (V and ⁇ ). and Trx6 rrx-transfected WEHI7.2 cells (D and S Twenty mice were injected s-c. with 2 X 10 ⁇ cells and 0 I ml matngel in the flank. Tumor size was measured every 3-4 days with calipers, and tumor volumes were calculated. Nine days after tumor cell injection, half of the mice were injected %x. into the opposite flank with I m( ⁇ g/day dexamethasone tO. V. and D) or with vehicle alone (•. ⁇ .
  • 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 rrx-transfected cells were resistant to apoptosis induced by dexamethasone Trx has been reported to be necessary for assembly of the glucocorticoid receptor (8) However, glucocorticoid 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 rrx-transfected cells also showed resistance to apoptosis induced by staurosporine. etoposide. ⁇ /-acetyl-sph ⁇ ngos ⁇ ne.
  • Bcl-2 is believed to exert its inhibitory effects upstream of the activation of the cysteine aspartate proteases cascade (caspase) responsible for the final stages of apoptosis (28)
  • the protective effects of Bcl-2 against apoptosis have been suggested to involve an amioxidam mechanism (291.
  • Trx a gene that codes for a known redox-active protein, also inhibits apoptosis
  • WEHI7 2 cells transfected with trx formed tumors in scid mice that grew, considerably faster than tumors formed by the wild-type parental cells or bv r «7-2-transfec ⁇ ed cells This may be due. in pan.
  • Trx system offers a novel target for agents to promote apoptosis and inhibit rumor growth, as well as to reverse the drug resistance of some cancers It is interesting, therefore, that some 2- ⁇ m ⁇ dazolyl disulfide inhibitors of Trx (37) have been shown to induce apoptosis in cancer cells (38) and, in animal studies, to have antitumor effects (38)
  • transfection with 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 rrx could be a new human protc-oncogene.
  • Lam. M Dubyak. G . Chen. L . Nunez. G Miesfeld. R L . and Distelhorsi. C W Evidence that BCL-2 represses apoptosis bv regulating endoplasmic reticulum-assc- c ⁇ a ⁇ ed Ca * fluxes Proc Nail Acad Sci USA. 9/ 6569-6573. 1994 Rurxllett. S E-. Gordon. D A., and Miesfeld. R L Characte ⁇ zation of a panel of rat ventral prostate epithelial cell lines immortalized in Ihe presence or absence of androgens Exp Cell Res.. 203 214-221 1992
  • sotnera.se inhibitors induce apoptosis in thymocytes. Biochim. Biopftys. Acta. 1175: 147-154. 1993.
  • Trolox inhibits peroxynitriie-mediated oxidative stress and apoptosis in rat thymyocytes. Arch. Biochem. Bi ⁇ phys.. 333: 482-488. 19%.
  • 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 cof actor for ribonucleotide reductase, the first unique step in DNA synthesis (4). More recently thioredoxin has been shown to exert redox controi over a number of transcription factors, including NF-KB (5), TTIHC (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 fibroblasts 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 (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 Assav Apoptotic cells were detected utilizing the TUNEL assay (23, 24) adapted to an automated in situ hybridization instrument (gen IT, Ventana Medical Systems, Inc.).
  • the TUNEL assay utilizes recombinant terminal deoxynucleotidyl transferase (Tdt) (GIBCO 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
  • Biotin 16-dUTP Biotin 16-dUTP
  • 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).

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

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US12/694,577 US20100166724A1 (en) 1996-12-06 2010-01-27 Uses of thioredoxin

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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
CN1302107C (zh) 1998-01-30 2007-02-28 吉倪塞思技术公司 与硫氧还蛋白或硫氧还蛋白还原酶基因互补的寡核苷酸序列以及将其用于调节细胞生长的方法
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