Adult T-cell Leukemia-derived Factor Research Articles

Induction of ADF/TRX by oxidative stress in keratinocytes and lymphoid cells

Adult T-cell leukemia-derived factor (ADF)/human thioredoxin (TRX) has thiol-dependent reducing activities and is known to have regulatory roles on the DNA-protein interaction and cell activation. Inducive effect of ultraviolet (UV) has been indicated because of the enhanced expression of ADF/TRX in epidermal cells of sun-exposed skin, as determined by immunohistochemical staining with antibody against recombinant ADF (rADF). We studied the effect of UVB irradiation and other oxidative stress on the expression of ADF/TRX in epithelial cells as well as lymphoid cells, as HTLV-1 and Epstein-Barr virus-transformed lymphoid cells constitutively produce ADF/TRX. Using immunohistochemical staining anti-ADF antibody, the enhancement of ADF/TRX expression on primary culture of human keratinocytes was demonstrated, 12 h after 20 mJ/cm 2 UVB irradiation. Western blot analysis of the ADF/TRX protein in the cell lysates also showed the significant induction. In in situ hybridization, induction of ADF/TRX mRNA was detected after 4 h of UV exposure. ADF/TRX was also induced in a HTLV-1 (+) T-cell line, MT-1, by UVB or H 2O 2 dose dependently. The augmentation of ADF/TRX was observed 6 h after treatment of H 2O 2.

Inhibition of reperfusion injury by human thioredoxin (adult T-cell leukemia-derived factor) in canine lung transplantation

Human thioredoxin, which was previously recognized as adult T-cell leukemia-derived factor, has many physiologic activities, one of which is a radical scavenger effect. Its ability to reduce reperfusion injury was assessed in vivo in a canine lung transplantation model. In 19 dogs, left lung allotransplantation was performed after 100 minutes of warm ischemia. The function of the transplanted lung was assessed after clamping of the contralateral pulmonary artery. In the human thioredoxin group (n = 6), human thioredoxin 30 mg/kg was given to the recipients during reperfusion. In the N-acetylcysteine group (n = 5), N-acetylcysteine 150 mg/kg, known as a radical scavenger, was given in the same manner. In both groups, arterial oxygen tension was significantly higher than in the control group (n = 8). In the human thioredoxin group, peak inspiratory pressure was significantly lower than in the control group. Macroscopic and microscopic examinations showed an almost normal appearance of the lung tissues in the human thioredoxin and N-acetylcysteine groups, in contrast to the abnormal findings in the control group. Thus it would appear that human thioredoxin has a protective effect on transplanted lungs, as does N-acetylcysteine, and that its action may be a radical scavenger effect.

Adult T cell leukemia-derived factor/human thioredoxin protects endothelial F-2 cell injury caused by activated neutrophils or hydrogen peroxide

Adult T cell leukemia-derived factor (ADF), originally defined as an interleukin 2 receptor/alpha (alpha) chain inducer produced by human T-lymphotropic virus type-I transformed cells, is identical to human thioredoxin (TRX). In this study, the protective effect of ADF/TRX on the cytotoxicity of endothelial cells caused by phorbol myristate acetate (PMA)-activated neutrophils or hydrogen peroxide (H2O2) was examined. When murine endothelial F-2 cells established from an ultraviolet light-induced tumor on a nude mouse were incubated with PMA-activated neutrophils or with 1 mM H2O2 for 6 hours, the cytotoxicity of F-2 cells was respectively 51 +/- 4% or 40 +/- 8% by the 51Cr releasing assay. Recombinant ADF/TRX (rADF/TRX) inhibited this cytotoxicity in a dose-dependent manner, although mutant ADF/TRX (cysteine 31 to serine), 2-mercaptoethanol and dithiothreitol did not. On a molar basis, rADF/TRX was more effective than glutathione but less effective than catalase. Immunoblotting analysis showed that treatment with 0.1 mM H2O2 induced murine TRX on F-2 cells. These findings indicate that ADF/TRX is an oxidative stress-inducible endogenous protein and rADF/TRX plays a protective role against activated neutrophils- or H2O2-induced endothelial cytotoxicity.

Neuroprotection by glial cells through adult T cell leukemia-derived factor/human thioredoxin (ADF/TRX)

Adult T cell leukemia-derived factor (ADF) is a human homologue of thioredoxin (TRX) with many biological functions and is induced by various stimuli and stress. In the central nervous system (CNS), expression of ADF/TRX occurs in glial cells during ischemia and reperfusion. We showed that ADF/TRX was actively released from U251 astrocytoma cells upon exposure to a low concentration of H 2O 2. The addition of conditioned medium from H 2O 2-stimulated U251 cells or recombinant ADF (rADF) to the culture medium promoted the survival of neurons from embryonic mouse cortex and striatum, but the addition of mutant ADF (mADF), which has no reducing activity, did not. In addition to rADF, incubation with two other thiol compounds, 2-mercaptoethanol (2-ME) andN-acetyl- l-cysteine (NAC), also increased the neuronal cell survival rate. In contrast, l-buthionine-(S,R)-sulfoximine (BSO), which inhibited the synthesis of glutathione (GSH), decreased the neuronal cell survival rate. Intracellular GSH was increased by incubation with rADF for 24 h, as it is with 2-ME and NAC. Redox active molecules such as thiol compounds may be survival factors for central neurons in vitro, and this capacity may be supplied by endogenous molecules, such as ADF/TRX and glutathione, under certain pathologic conditions in vivo.

Detection of adult T-cell leukemia-derived factor/human thioredoxin in human serum

Adult T-cell leukemia (ATL)-derived factor (ADF), originally defined as an inducer of interleukin-2 receptor/α-chain (IL-2R/p55) of human T-lymphotropic virus type I (HTLV-I) positive T cells, is a human homologue of redox-active coenzyme thioredoxin (Trx) of Escherichia coli. In this study, an enzymatic assay system based on the dithiol-dependent insulin-reducing activity of ADF/Trx was established (insulin-reducing assay) to determine the amount of ADF/Trx in human serum using NADPH and Trx reductase purified from human placenta. Insulin-reducing activity was detected in all of the serum samples from healthy volunteers ( n = 30) screened by this assay, with a mean ±SD of 10.9 ± 2.4 U/l. This mean value corresponds with the concentration of 223 ng recombinant ADF/Trx (rADF/Trx)/ml. Human serum is known to contain several redox-active proteins with ADF/Trx motifs. To differentiate the contribution of these proteins and ADF/Trx to the insulin-reducing activity, the anti-rADF/Trx monoclonal antibody (mAb)-conjugated affinity column-depleted sera obtained from an identical source was used for analysis. The affinity column-depleted sera demonstrated a loss of over 99% of the original activity, while control column depleted sera lost less than 4%. Furthermore, the amount of affinity-purified ADF/Trx molecules eluted from the same column almost corresponded with the amount estimated by the insulin-reducing activity. These results showed the presence of ADF/Trx detected by the insulin-reducing assay in human serum and the affinity purification with specific anti-ADF/Trx mAb, indicating the possible role of serum ADF/Trx as a systemic redox regulator and/or as a radical scavenger.

EFFECT OF ESCHERICHIA-COLI THIOREDOXIN, A HOMOLOG TO THE ADULT T-CELL LEUKEMIA-DERIVED FACTOR, ON EPSTEIN-BARR VIRUS-TRANSFORMED B-LYMPHOCYTES

In order to investigate the role of autocrine growth factors on cell growth of Epstein-Barr virus (EBV)-transformed human B lymphocytes, we have studied the effect of E. coli recombinant thioredoxin (r-thioredoxin), a homologue of adult-T-cell leukemia-derived factor (ADF), on the cell growth of various human lymphoid cell lines, either EBV-positive or -negative. It was found that, in serum-free culture conditions, exogenous r-thioredoxin increases DNA synthesis in lymphoblastoid and Burkitt's cell lines, independently of EBV genome expression; moreover, the combined effect of r-thioredoxin and interleukin-I (IL-1) or IL-6 resulted in a marked increase in DNA synthesis in lymphoblastoid cells, but not in Burkitt's cell lines. Anti r-thioredoxin monoclonal antibodies were developed and used to test the possibility of interfering with r-thioredoxin-induced cell proliferation. It was found that the growth-promoting activity of r-thioredoxin was inhibited by an anti-r-thioredoxin monoclonal antibody in a dose-dependent manner in P3HR-1 cells, a Burkitt's lymphoma cell line harboring an EBNA-2-defective virus, but not in other lymphoid cell lines, thus indicating that target cells may play an active role in antibody-mediated thioredoxin neutralization.

Thiol-mediated redox regulation of lymphocyte proliferation. Possible involvement of adult T cell leukemia-derived factor and glutathione in transferrin receptor expression.

The proliferative response of PBMC to PHA, Con A, OKT3 mAb and IL-2-dependent proliferation of PHA-blasts was examined in a thiol-free environment (cultured in a L-cystine- and GSH-free medium). [3H]TdR incorporation assay and cell cycle analysis revealed that stimulated PBMC could not enter the S phase when deprived of these thiol compounds. In thiol-free cultures, an increase in intracellular free Ca2+ concentration and IL-2R alpha-chain/p 55 (Tac) induction was still observed, whereas transferrin receptor induction was markedly reduced, suggesting that the proliferative response of mitogenically stimulated PBMC was arrested in the late G1 phase in which transferrin receptor is induced. In GSH-depleted cultures, a similar reduction of the proliferative response of PBMC and PHA-blasts was observed when the concentration of L-cystine was lowered, in a dose-dependent manner. Each reduction or loss of proliferative response was partially restored by supplementation of 2-ME or adult T cell leukemia-derived factor (ADF)/human thioredoxin which is considered to be an endogenous dithiol-reducing factor. L-Cystine transport analysis showed that mitogenically stimulated PBMC and PHA blasts incorporated L-cystine, whereas resting PBMC did not. Furthermore, ADF as well as 2-ME exhibited an enhancing activity on the L-cystine transport in PHA blasts. Together with the fact that L-cystine transport is a limiting step in glutathione synthesis, these findings suggest that GSH and ADF might cooperate in the thiol-mediated redox regulation process and might also play key roles in cell cycle (late G1 to S) progression of activated lymphocytes.

Mouse Thioredoxin Gene Maps on Chromosome 4, whereas Its Pseudogene Maps on Chromosome 1

Thioredoxins are involved in various biochemical systems and mediate both redox and nonredox functions. In mammalian cells, thioredoxin functions as an endogenous glucocorticoid receptor activating factor and as an adult T-cell leukemia-derived factor (ADF) that stimulates expression of interleukin-2 receptor in human T-cell leukemia virus (HTLV)-I transformed T cells. We have mapped the thioredoxin gene ( Txn) and its processed pseudogene ( Txn-ps1) in the mouse using a panel of interspecific backcross mice. Txn maps to Chr 4, whereas Txn-ps1 maps to the proximal region of Chr 1.

Dysregulation of adult T-cell leukemia-derived factor (ADF)/thioredoxin in HIV infection: loss of ADF high-producer cells in lymphoid tissues of AIDS patients.

Adult T-cell leukemia (ATL)-derived factor (ADF) is a multifunctional protein homologous to thioredoxin (TRX) with co-cytokine and thiol-dependent reducing activities. ADF/thioredoxin production is enhanced in T cells transformed by HTLV-I. We have examined the effect of HIV-1 infection on ADF/TRX expression using specific antibody against ADF/TRX. Lymph nodes from 5 AIDS and 1 AIDS-related complex (ARC) patients were examined. As a control, 8 HIV noninfected lymph nodes, including 3 cases with hyperplasia, were also examined. Immunohistopathological studies using normal HIV noninfected lymph nodes showed that ADF/TRX high-producer (ADFh) cells were macrophages and cells with dendritic morphology in the paracortical area. Abundant ADFh cells were observed in HIV noninfected hyperplastic lymph nodes. The number of ADFh cells was low in hyperplastic lymph nodes from an ARC patient. All of the lymph nodes of 5 AIDS cases were atrophic and the number of ADFh cells were extremely low. To verify these histochemical studies, we examined the effect of in vitro HIV infection on ADF/TRX expression in HTLV-I (+) T-cell lines. Western blot analysis showed that a reduction of ADF/TRX in HIV-1-infected SKT-1B and MT-2 cells, and the reduction inversely correlated with p24 antigen level. On the basis of the above in vivo and in vitro findings, we imply that the levels of ADF/TRX were down-regulated by HIV-1 infection and that the down-regulation may play a role for pathophysiology of HIV-infected individuals.

Reactive oxygen-reducing and protein-refolding activities of adult t cell leukemia-derived factor /human thioredoxin

Reducing and protein-refolding activities of adult T cell leukemia-derived factor (ADF)/human thioredoxin were studied. Recombinant ADF/human thioredoxin produced by E. coli, which has an insulin-reducing activity as efficient as that of E. coli thioredoxin, also reduced some reactive oxygen species, such as hydrogen peroxide. Furthermore, recombinant ADF/human thioredoxin was found to have protein-refolding activity for scrambled (mispaired disulfide-containing) RNase A. Cys-31 at the active site of ADF/human thioredoxin proved essential for reducing activity, and loss of Cys-31 in ADF/human thioredoxin attenuated the protein-refolding activity. These data suggest a physiological role of ADF/human thioredoxin in protecting living cells from proteotoxicity caused by reactive oxygens in vivo.

Immunohistochemical Distribution of Adult T-Cell Leukemia-Derived Factor/Thioredoxin in Epithelial Components of Normal and Pathologic Human Skin Conditions

Tissues from normal human skin and various skin diseases were studied with the immunoperoxidase technique using an antibody to adult T-cell leukemia-derived factor (ADF), a homologue of human thioredoxin. Normal human skin showed positive immunostaining for ADF/thioredoxin in the outer root sheath of hair follicle, sebaceous glands, and secreting components of apocrine and eccrine sweat units, but not in the unexposed interfollicular epidermis and other parts of both hair follicles and the sweat units. Immunoreactivity of benign skin tumors gave similar distribution to their normal counterparts; trichilemmal cyst, nevus sebaceus, senile sebaceous hyperplasia, and mixed cell tumor were positive for immunostaining, whereas epidermal cyst and pilomatricoma were not. No immunoreactivity was detected in malignant skin tumors such as basal cell carcinoma and poorly differentiated squamous cell carcinoma. Solar keratosis, well-differentiated squamous cell carcinoma, some of metastatic lesions of squamous cell carcinoma, and extramammary Paget's disease reacted with the antibody. These immunoreactivities reflected numerous functions of thioredoxin in higher organisms. Our findings suggest that the expression of ADF/thioredoxin in both normal and abnormal human skin is related to epithelial cell differentiation.

Expression and growth-promoting effect of adult T-cell leukemia-derived factor. A human thioredoxin homologue in hepatocellular carcinoma.

Adult T-cell leukemia-derived factor (ADF), originally defined as an interleukin-2 receptor inducer, is a human thioredoxin homologue. ADF is detected in many malignant tissues and has a growth-promoting effect on transformed cells. In this study, ADF expression was examined immunohistochemically in human liver cell lines and liver tissues, and its growth-promoting effect was tested on human hepatoma cells. On three liver cell line--PLC/PRF/5, HepG2, and Chang liver cells--ADF stained positively and also was detected by immunoblotting. ADF had strong staining in the fetal liver (n = 8), although it was faint in the normal adult liver (n = 6). In hepatocellular carcinoma (n = 25), ADF expression generally was enhanced and was very strong in 52% (13 of 25) of the cases, although it was moderate in cases of chronic hepatitis or cirrhosis. ADF augmented the growth of PLC/PRF/5 cells and showed an additive effect with epidermal growth factor. These results indicate possible involvement of ADF in cell activation and growth of hepatocytes, as is the case with lymphocytes.

Lymphocyte transformation and thiol compounds: The role of ADF/thioredoxin as an endogenous reducing agent

ADF (adult T-cell leukemia-derived factor), an inducer of IL-2R with growth promoting activity, is a homologue of thioredoxin which is involved in many thiol-dependent reducing reactions. ADF is constitutively produced and released by human lymphoid cell lines transformed by lymphocyte-tropic viruses, such as human T-lymphotropic virus type I (HTLV-I) and Epstein-Barr virus (EBV). We found that the viability and growth of these ADF high-producer cell lines (ATL-2, HUT102, MT-2, 3B6 and RPMI8866) were highly dependent on l-cystine in the culture. In contrast to the relative cystine independency of ADF low-producer cells (Jurkat, Jijoye, U937 and K562), the growth of ADF high-producer cells was almost completely suppressed in l-cystine-free condition. Their viability and growth in l-cystine-free medium were markedly improved by 5 × 10 −5M l-cysteine, 5 × 10 −5 M 2-ME or 10 −3 M GSH and partially by 10 −3 M DTT. The results demonstrate the requirement of reducing condition involving thiol compounds for the optimal growth of the virally transformed lymphoid cells. Furthermore, recombinant ADF (rADF) and suboptimal dose of 2-ME additively enhanced the growth of ATL-2 cells in l-cystine-free medium, implying the possible involvement of endogenous reducing agents such as ADF/thioredoxin homologue in the process of lymphocyte transformation/activation.

Protective activity of adult T cell leukemia-derived factor (ADF) against tumor necrosis factor-dependent cytotoxicity on U937 cells.

Adult T cell leukemia-derived factor (ADF) is a human homologue of thioredoxin with many biologic functions including IL-2R induction, growth promotion, thiol-dependent reducing activity, and radical scavenging activity. The regulatory effect of ADF on the cytotoxic activity of TNF was examined by using a human histiocytic lymphoma cell line, U937. When U937 cells were preincubated with recombinant ADF (rADF) (0.1-100 micrograms/ml) at 37 degrees C for 30 min, TNF-dependent cytotoxicity on U937 cells was markedly inhibited. This inhibitory effect was as high as 95% in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (rADF 100 micrograms/ml) and 85% in the 51Cr-releasing assay (rADF 10 micrograms/ml). After pretreatment of U937 cells with IFN-gamma to augment the sensitivity to TNF, an inhibitory effect of rADF was also found. When U937 cells were washed after preincubation with rADF, resistance to TNF-dependent cytotoxicity was still observed, indicating that rADF inhibited the sensitivity of U937 to TNF-dependent cytotoxicity rather than modifying TNF molecules. Scatchard analysis of TNF receptors on U937 cells using 125I-TNF showed that rADF modulated neither the density nor the affinity of the cell membrane significantly. rADF also reduced the cytotoxicity induced by anti-Fas IgM mAb which shows cytotoxicity quite similar to TNF. rADF (10 micrograms/ml) reduced 90% of the cytotoxicity by anti-Fas IgM mAb, without a detectable change either in Fas Ag expression (MFI 58.1 vs 53.3) or in the degradation of anti-Fas IgM mAb as determined by flow cytometric analysis. These findings indicated that the rADF-induced resistance to the cytotoxic effect of TNF and anti-Fas mAb was not related to the modulation of the TNF receptor or Fas Ag.

Coexpression of adult T-cell leukemia-derived factor, a human thioredoxin homologue, and human papillomavirus DNA in neoplastic cervical squamous epithelium.

Adult T-cell leukemia-derived factor (ADF) is an autocrine interleukin-2 receptor-inducing factor produced by human T-lymphotropic virus-1 (HTLV-1)-transformed lymphocytes, which has a high structural homology with an endogenous dithiol reducing coenzyme, thioredoxin. Its localization was investigated immunohistochemically in the cervix, using normal tissue (27 samples) and squamous neoplastic tissue (three condylomas, 42 cervical intraepithelial neoplasia [CIN] samples, 34 invasive squamous cell carcinoma samples). The expression of human papillomavirus (HPV) DNA was also studied in serial sections of the same subjects. Normal squamous cells and glandular cells of the cervix were negative for ADF. However, intracytoplasmic and/or intranuclear ADF-positive cells were usually found in the intermediate and superficial layers of the neoplastic squamous epithelium of condylomas (three of three cases) and CIN (35/42 cases). HPV DNA was detected in all condylomas and in 27 of 42 CIN specimens. HPV DNA-positive cells were usually localized in the intermediate and superficial layers of the neoplastic squamous epithelium. These HPV DNA-positive cells were also positive for ADF. Invasive squamous cell carcinoma was also positive for ADF (24/34 cases) and HPV DNA (11/34 cases). The coexpression of HPV DNA and ADF was observed in all HPV DNA-positive cases. Coexistence of HPV DNA and ADF immunopositivity in neoplastic squamous cells of the cervix suggests that ADF expression closely reflects the intracellular event on HPV DNA replication.

Hepatocyte growth inhibitory factor derived from HTLV-I(+) T cell lines: Effect on the epidermal growth factor-dependent proliferation of rat hepatocytes

A human T cell leukemia virus-I infected T cell line, ATL-2, produces an interleukin-2 receptor inducing factor, adult T cell leukemia (ATL)-derived factor (ADF). In the conditioned medium (CM) of ATL-2, we found an inhibitory activity on the epidermal growth factor (EGF)-dependent proliferation of primary cultured rat hepatocytes, measured by cell number and [ 3H]thymidine incorporation. ATL-2 CM dose-dependently inhibited hepatocyte proliferation. This activity was fractionated by gel filtration at a molecular size of 15,000 to 40,000 and was tentatively called hepatocyte growth inhibitory factor (HGI). Further fractionation with the ion-exchange column indicated that HGI was separable from ADF. Nevertheless, there was a positive correlation between HGI and ADF production, because the HGI activity was also detected in the CM of another ADF producer cell line (HUT102), while no significant HGI activity was detected in the CM of low ADF producer cell lines, ED and MOLT4.

ADF, A Growth-Promoting Factor Derived from Adult T Cell Leukemia and Homologous to Thioredoxin: Involvement in Lymphocyte Immortalization by HTLV-I and EBV

Publisher Summary Adult T-cell leukemia-derived factor (ADF) plays an important role in the transforming properties of HTLV-I by interfering with the expression of the interleukin-2 receptor (IL-2R) complex. In particular, ADF appears to act as an autocrine growth factor for an Epstein–Barr virus (EBV)-positive B cell line established independently by another group and to synergize with suboptimal doses of other lymphokines, making these cells sensitive to a series of growth factors. The fact that both HTLV-I-transformed T lymphocytes and EBV-immortalized B cells are able to produce the same factor implies that some underlying mechanisms of transformation could be common to the two viruses, which could possibly interact with similar cellular genes. The amino acid sequence of ADF is highly homologous to prokaryotic and mammalian thioredoxins. Thus, ADF appears to be a member of the thioredoxin family of protein cofactors involved in thiol-dependent redox reactions in many cell types and at various steps of cellular metabolism. ADF secreted by the cells infected with HTLV-I or EBV exhibits a very high reducing potential. This enzymatic activity of ADF might, at least partly, explain its role in cellular activities, including IL-2R expression, increased susceptibility to a variety of growth factors, and promotion of cell proliferation.

Adult T-cell leukemia-derived factor/thioredoxin, produced by both human T-lymphotropic virus type I- and Epstein-Barr virus-transformed lymphocytes, acts as an autocrine growth factor and synergizes with interleukin 1 and interleukin 2.

Interleukin 1 (IL-1) has been obtained from the Epstein-Barr virus-infected B-lymphoblastoid cell line 3B6 and shown to be involved in autocrine growth of 3B6 B cells. Independently, adult T-cell leukemia-derived factor (ADF) was purified from human T-lymphotropic virus I-infected leukemic T-cell line (ATL-2) and reported as an interleukin 2 (IL-2) receptor-inducing factor. We have previously reported the same molecular mass, pI, and NH2-terminal amino acid sequence for both 3B6-derived IL-1 and ADF. cDNA cloning of ADF demonstrated high homology with the prokaryotic disulfide reducing enzyme thioredoxin. We show here that ADF and 3B6-derived IL-1 are identical. By RNA blot, 3B6 and ATL-2 cells were shown to contain high levels of 0.6-kilobase mRNA corresponding to ADF. Such message was not detected in resting peripheral blood lymphocytes but could be weakly induced by lymphocyte activation. Antibodies have been raised against synthetic peptides corresponding to the NH2 terminus and the COOH terminus of ADF. Immunoblotting and sequential immunoprecipitation with these antibodies revealed the same 13-kDa protein in 3B6 and ATL-2 cells. Recombinant ADF could sustain growth of 3B6 and ATL-2 cells at low cellular concentration without fetal calf serum; ADF, thus, appears involved in their autocrine growth. Similarly, recombinant ADF could enhance growth of other B-cell lines, including the Epstein-Barr virus-negative Burkitt lymphoma line BL41 and the lymphoblastoid cell lines CRAG8, CRB95, and 1G8. Finally, recombinant ADF exhibits marked synergism with other cytokines, such as IL-1 and IL-2, allowing virally infected lymphocytes to respond to suboptimal amounts of a variety of growth factors.