Human T-cell leukemia virus-1-positive cell line established from a patient with small cell lung cancer.

A stable cell line, KHM-3S, was established from a patient with small cell lung cancer (SCLC), who had a high serum level of soluble interleukin 2 receptors (sIL2-R) and was seropositive for human T cell leukemia virus (HTLV)-1. KHM-3S cells were positive for IL2-R (Tac) and NKH-1, but negative for other lymphocytic markers such as OKT 11, OKT 4, OKT 8, T cell receptor (WT 31), B 1, and B 4. Moreover, the KHM-3S cells were negative for leukocyte common antigen and strongly positive for neuron-specific enolase (NSE). Secretion of sIL2-R and NSE by the KHM-3S line was detected by an enzyme-linked immunosorbent assay. Rearrangement of the T cell receptor gene and monoclonal HTLV-1 integration were found by Southern blot analysis of KHM-3S DNA. However, Northern blot analysis showed no T cell receptor mRNA. KHM-3S may be useful for studies on the role of HTLV-1 in carcinogenesis and IL2-R expression in SCLC.     

A unique T-cell line derived from an HTLV-1-negative adult T-cell leukemia patient

A unique T-cell line, designated ATL-5T, was established from lymphoma cells in pericardial effusion of an adult T-cell leukemia (ATL) patient not carrying HTLV-1 provirus. The cell line is OKT4 and/or Leu3a+ and OKT8 and/or Leu2a+, but interleukin 2 receptor (IL2R)- and HTLV-1 provirus genome negative, and has cytogenetically abnormal karyotypes. The cell line contains rearranged T-cell receptor beta-chain gene, which was identical in rearrangement pattern to the T-cell receptor beta-chain gene in primary cells. These results suggest that factors other than HTLV-1 may sometimes be associated with HTLV-1-negative ATL. The ATL-5T cell line we describe here is unique, and should contribute to further elucidation of the mechanisms involved in the pathogenesis of HTLV-1-negative ATL and HTLV-1-positive ATL.     

Human T-cell leukemia virus type 1 associated with small cell lung cancer

A patient with small cell lung cancer (SCLC) whose serum contained high levels of soluble interleukin-2 receptors is reported. Soluble interleukin-2 receptors in the supernatant of cultured SCLC cells obtained from the patient's pleural effusion while he had malignant pleuritis, increased almost linearly from the time of cell seeding. The expression of interleukin-2 receptors (Tac) on the SCLC cells were demonstrated by an immunofluorescence study. However, other lymphocytic markers, including OKT 11, OKT 4, OKT 8, B 1, and B 4, were not found on the cells with the exception of the natural killer cell marker, NKH-1. Southern blot analysis indicated the rearrangement of the T-cell receptor of the cancer cells. Moreover, monoclonal integration of human T-cell leukemia virus type 1 (HTLV-1) provirus in DNA from the cancer cells was also demonstrated. These observations suggest that some SCLC in HTLV 1 endemic areas are associated with HTLV-1.     

Expanded Tγ cell populations with the morphology of large granular lymphocytes — I. Immunological,clinical and morphological characterization

Lymphocytes from two patients with Tγ cell proliferations displaying the morphology of large granular lymphocytes (LGL) were characterized in terms of cell marker phenotyping and immunologic functions. In both patients, the lymphocytes were positive for E-R, HuTLA, OKT5, OKT8, OKT11, OKM1, VEP13, Leu1, Leu2a, Lyt2 and Lyt3 and were negative for Tμ, Tar, SIg, BA1, BA2, E_M-R, C₃d-R, C₃b-R, OKT6, OKT9, Leu3a, OKIa1 and TdT. In addition, investigations for T411, T811 and M522 in patient 1 yielded positive results. There were differences in the phenotype of the two patients with regard to the reactions with OKT3, OKT10 and VEP10. While, in patient 1, OKT3 was very pronounced and OKT10 and VEP10 were completely negative, OKT10 and VEP10 were very pronounced in patient 2, whereas OKT3 was positive only in a very small percentage of cells. Though the lymphocytes in both patients were potent effectors of NK and K functions (patient 2 more strongly than patient 1 and a noticeably reduced mitogen response was shown to PHA, Con A and zinc, patient 1 showed a distinct suppression of allogenic and autologous B cell response to transformation into ISC, coinciding with the clinical observation of a hypogammaglobulinemia; neither B cell suppression nor dysgammaglobulinemia was seen in patient 2. The results are discussed with regard to other comparable Tγ proliferations reported in the literature.     

Immunoprecipitation of the interleukin-2 receptor from Hodgkin's disease derived cell lines by monoclonal antibodies

The nature of Hodgkin and Reed-Sternberg (H-RS) cells and their normal counterpart remains a matter of controversy. Our recent investigations have suggested that H-RS-cells derive from certain activated lymphocytes of either B or T cell origin. In keeping with this concept, we were able to demonstrate that in the majority of cases of Hodgkin's disease the interleukin-2 receptor (IL2-R) was detectable on H-RS-cells by three monoclonal antibodies (anti-Tac, Tü69 and ACT-1) with the sensitive alkaline phosphatase anti-alkaline phosphatase (APAAP) tissue staining procedure. In extension of these studies, we precipitated IL2-R antigen from two established permanent cell lines (L540 and L591) derived from patients with Hodgkin's disease. Although these cell lines are different in nature, in that the L540 line shows rearrangement for the T cell receptor beta chain gene and the L591 line displays rearrangement for immunoglobulin genes (gamma heavy and lambda light chain), the antigen precipitated by monoclonal antibodies against IL2-R exhibited a molecular weight of approximately 53 kd on the L591 line and 58 kd on the L540 line. Thus, these data strongly support the view that the labelling of H-RS-cells in tissue sections for IL2-R was not merely due to cross-reactivities of the two anti-IL2-R antibodies with a determinant of an unrelated structure but represents true IL2-R molecules.     

食管小细胞癌与小细胞肺癌凋亡生物学特性的研究

[目的]探讨原发性食管小细胞癌(primary esophageal small cell carcinoma,PESC)和小细胞肺癌(small cell lung carcinoma,SCLC)凋亡生物学特性。[方法]采用免疫组织化学SP法检测survivin,caspase-3,bcl-2,mtp53在PESC和SCLC中的表达。[结果]PESC和SCLC中survivin,caspase-3,bcl-2,mtp534种蛋白的阳性分布无统计学差异(P>0.05)。在PESC和SCLC survivin阴性组中,caspase-3阳性表达率分别为72.41%和62.07%,均明显高于survivin阳性组(30.00%和25.00%,P<0.05)。在这两种小细胞癌中,survivin与caspase-3表达呈负相关;bcl-2与mtp53的表达呈正相关;bcl-2与caspase-3或survivin之间无相关性。[结论]食管小细胞癌和小细胞肺癌的抗凋亡通路存在诸多相同之处,survivin,bcl-2和mtp53均参与了两种小细胞癌的发生发展。     

Growth of a cultured leukemia subline was promoted by conditioned medium of thymic reticuloepithelial-like cells (B6TE)

A murine leukemia subline (L17R) was selectively developed in the presence of conditioned medium of a thymic reticuloepithelial-like cell line (B6TE). Cytotoxicity tests and immunofluorescence microscopy showed that L17R cells were negative in the expression of Thy 1.1, Lyt 1.2 and terminal deoxynucleotidyl transferase (TdT), however, 35% positive in Lyt 2.1 phenotype, and 95% positive in the expression of peanut agglutinin (PNA) receptor. B6TE conditioned medium had no activity of interleukin 1 (IL 1), interleukin 2 (IL 2), interleukin 3 (IL 3) and granulocyte/macrophage colony-stimulating factor (GM-CSF). When L17R leukemic cells were plated at a low cell density, their growth was accelerated 40 times by the addition of concentrated B6TE culture supernatant. This growth activity, tentatively designated leukemia-growth-promoting factor (LGPF), was heat sensitive, and its mol. wt was estimated to be approx. 25,000 from the elution pattern of Sephadex G-100 chromatography.     

Downstream signalling and specific inhibition of c-MET/HGF pathway in small cell lung cancer: implications for tumour invasion

The c-MET receptor can be overexpressed, amplified, or mutated in solid tumours including small cell lung cancer (SCLC). In c-MET-overexpressing SCLC cell line NCI-H69, hepatocyte growth factor (HGF) dramatically induced c-MET phosphorylation at phosphoepitopes pY1230/1234/1235 (catalytic tyrosine kinase), pY1003 (juxtamembrane), and also of paxillin at pY31 (CRKL-binding site). We utilised a global proteomics phosphoantibody array approach to identify further c-MET/HGF signal transduction intermediates in SCLC. Strong HGF induction of specific phosphorylation sites in phosphoproteins involved in c-MET/HGF signal transduction was detected, namely adducin-alpha [S724], adducin-gamma [S662], CREB [S133], ERK1 [T185/Y187], ERK1/2 [T202/Y204], ERK2 [T185/Y187], MAPKK (MEK) 1/2 [S221/S225], MAPKK (MEK) 3/6 [S189/S207], RB [S612], RB1 [S780], JNK [T183/Y185], STAT3 [S727], focal adhesion kinase (FAK) [Y576/S722/S910], p38alpha-MAPK [T180/Y182], and AKT1[S473] and [T308]. Conversely, inhibition of phosphorylation by HGF in protein kinase C (PKC), protein kinase R (PKR), and also CDK1 was identified. Phosphoantibody-based immunohistochemical analysis of SCLC tumour tissue and microarray established the role of c-MET in SCLC biology. This supports a role of c-MET activation in tumour invasive front in the tumour progression and invasion involving FAK and AKT downstream. The c-MET serves as an attractive therapeutic target in SCLC, as shown through small interfering RNA (siRNA) and selective prototype c-MET inhibitor SU11274, inhibiting the phosphorylation of c-MET itself and its downstream molecules such as AKT, S6 kinase, and ERK1/2. Investigation of mechanisms of invasion and, ultimately, metastasis in SCLC would be very useful with these signal transduction molecules.     

Transformation of different phenotypic types of human bone marrow T-lymphocytes by htlv-1

Fresh bone marrow mononuclear leukocytes were used as a target for infection by human T-cell leukemia-lymphoma virus subgroup 1 (HTLV-1) by co-cultivation with virus-positive cell lines. The lines were established from T-cell leukemia-lymphoma patients or HTLV-1-transformed human umbilical cord-blood T cells. Clumps of transformed cells became visible by 2–3 weeks after infection and developed at a high frequency (>90%) in the bone marrow samples used. Stimulation of target cells with lymphocyte mitogens facilitated this process but was not absolutely required. Unlike fresh or cultured cells from HTLV-1-positive adult leukemia-lymphoma patients and HTLV-1-transformed cord-blood T cells, which usually have an OKT 4/leu 3a surface phenotype, the transformed bone marrow cells frequently fell into one of three categories based on their reactivity with cell-specific monoclonal antibodies. These included populations of cells that were predominantly; (1) OKT 4/leu 3a-positive, (2) OKT 8/leu 2a-positive, and (3) cells expressing neither phenotype. Fresh bone marrow provided a rapid and efficient system for the assessment of HTLV-1 infection. The type of bone marrow cells transformed in vitro suggests that HTLV-1 can infect several subsets of T lymphocytes, possibly including immature cells or that these cells can undergo phenotypic modulation.     

Knockdown of Bcl-3 Inhibits Cell Growth and Induces DNA Damage in HTLV-1-infected Cells)

Oncoprotein Bcl-3 is perceived as an unusual member of IκB family since it can both stimulate and suppressNF-κB activation. Aberrant Bcl-3 results in increased cell proliferation and survival, suggesting a contributionto malignant potential and elevated levels of Bcl-3 have been observed in many HTLV-1-infected T cell lines andATL cells. To investigate the specific roles of Bcl-3 in HTLV-1-infected cells, we knocked down Bcl-3 expressionusing shRNA and then examined the consequences with regard to DNA damage and cell proliferation, as wellas NF-κB activation. The HTLV-1 encoded protein Tax promotes Bcl-3 expression and nuclear translocation.In HTLV-1-infected cells, Bcl-3 knockdown obviously induced DNA damage. Cell growth and NF-κB activationwere reduced in HTLV-1-infected or Tax positive cells when Bcl-3 expression was decreased. Together, our resultsrevealed positive roles of Bcl-3 in DNA stabilization, growth and NF-κB activation in HTLV-1-infected cells.     

Genetic analysis of amylase-producing cell lines: ectopic activation of the amylase gene by translocation.

Two amylase-producing cell lines have been established, KMK-2 from a patient with gastric cancer, and KHM-1B from a patient with IgA lambda-type multiple myeloma. Both patients exhibited extremely high levels of serum amylase. The production of S-type amylase m-RNA by KMK-2 and KHM-1B was demonstrated by Northern blot analysis. Chromosome analysis showed many qualitative and quantitative abnormalities in both cell lines. In KHM-1B, a translocation was found between 1p13 or 21, near the amylase genes locus, and 9q34, the abl oncogene locus. These findings suggest the amylase gene in KHM-1B to be activated by translocation. A rearranged amylase gene was demonstrated by Southern blot analysis with only one enzyme, Accl.     

Biological and Clinical Implication of Neuron-Specific Enolase and Creatine Kinase BB in Small Cell Lung Cancer

The specificity of neuron-specific enolase (NSE) and creatinekinase BB (CK-BB) for small cell lung cancer (SCLC) was determinedby biological and immunohistochemical procedures in lung cancertissues and cultured cell lines. Average values of extractableNSE and CK-BB of SCLC tissues were significantly higher thanthose of non-SCLC and normal lung tissues. A large amount ofNSE and CK-BB was demonstrated in SCLC cell lines. Immunohistochemical examination showed positive staining forNSE and CK-BB in most cases of SCLC and in a few cases of non-SCLC.From these data NSE and CK-BB should be considered to be highlyspecific for SCLC. In a clinical study serum values exceeding 10 ng/ml for NSEand 1.5 ng/ml for CK-BB were set as positive for the enzymes.Positive rates in SCLC were 71.4% for NSE and 65.3% for CK-BB,which were significantly higher than those in non-SCLC. Allpositive cases were in an advanced stage. Consecutive dailyNSE determinations during induction chemotherapy showed transientelevation immediately after the initiation of drug administration(tumor lysis syndrome), followed by a decline to the normalrange in responders. This phe nomenon seems to indicate tumorsensitivity to cytotoxic drugs. NSE positive non-SCLC was assensitive to cytotoxic drugs as SCLC. These findings indicatethat lung cancer with elevated serum NSE and CK-BB levels atdiagnosis should be strongly suspected of being SCLC in theadvanced stage.     

Prognostic value of serum soluble interleukin‐23 receptor and related T‐helper 17 cell cytokines in non‐small cell lung carcinoma

The signaling of interleukin (IL)‐23 and its receptor (IL‐23R) play a crucial role in the development of cancers. However, the clinical significance of human serum soluble IL‐23R (sIL‐23R) and its relationship with IL‐23 are still not explored in non‐small cell lung cancer (NSCLC). In our study, sIL‐23R was first identified in the serum of NSCLC patients, but not in healthy controls, by proteomics. The IL‐23R mRNA and protein were upregulated in NSCLC cell lines and tissues tested by quantitative PCR, western blot analysis and immunohistochemistry. The levels of sIL‐23R, IL‐23, and IL‐17 in 195 NSCLC patients’ serum were determined by ELISA, and high levels of sIL‐23R were significantly associated with advanced N stage (P = .039), clinical stage (P = .007), and poor 5‐year survival rate. In vitro, sIL‐23R was shown binding to IL‐23 and the balance could affect patients’ N and T stage, overall survival, and downstream cytokine IL‐17 in a potential antagonistic relationship. Although sIL‐23R, IL‐23, and IL‐17 were all associated with poor prognosis, only the sIL‐23R/IL‐23 ratio (hazard ratio, 1.945; 95% confidence interval, 1.147‐3.299; P = .014) was found to be an independent factor for prognosis. Therefore, we identified fragments of soluble cytokine receptor of IL‐23R with affinity ability to its natural ligand IL‐23 in NSCLC patients’ serum. The balance between the 2 antagonists can work as a potential prognostic serum marker.     

Transformation of human leukocytes by co-cultivation with HTLV-1-associated myelopathy patients' leukocytes

Sera and cerebrospinal fluid (CSF) from patients with human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy (HAM) were analyzed by Western blotting, and normal human leukocytes were transformed by co-cultivation with HAM patients' leukocytes. The sera and CSF from all HAM patients formed specific bands with HTLV-1 viral proteins, including p19, p24, p28, p32, p40 and p53. After 2-3 weeks of co-cultivation, scattered foci of cell aggregates were noted on macrophage sheets. Surface markers of the transformed cells were OKT3(+), OKT4(+), OKT8(-), IL-2 receptor(+) and EBNA(-). Chromosome analysis showed a normal karyotype. HTLV-1 viral genome was integrated into DNA isolated from transformed cell lines. Electron microscopy revealed type C virus particles in transformed T-cell lines. These results indicate that peripheral leukocytes from HAM patients can transform HTLV-1-negative leukocytes and HAM patients have the potential to acquire adult T-cell leukemia in the future.     

Activation of mTOR by human T-cell leukemia virus type 1 Tax is important for the transformation of mouse T cells to interleukin-2-independent growth

Human T-cell leukemia virus type 1 (HTLV-1) is a causative agent of adult T-cell leukemia, and it immortalizes and transforms human T cells in both an interleukin (IL)-2-dependent and -independent manner. HTLV-1 encodes Tax, which plays crucial roles in HTLV-1-mediated immortalization and transformation of human T cells. A previous study showed that Tax can transform a mouse T-cell line, CTLL-2, from having IL-2-dependent growth to IL-2-independent growth. Given that the Akt/mTOR pathway is essential for IL-2-induced cell growth in T cells, we examined whether the Akt/mTOR pathway is involved in Tax-induced transformation to IL-2-independent growth. The stable and transient expression of Tax in CTLL-2 induced the phosphorylation of p70S6 kinase and ribosomal protein S6, downstream targets of the mTOR kinase, whereas that of Akt was only minimally induced. Studies with Tax mutants indicated that the activation of mTOR by Tax was correlated with the transformation of CTLL-2 cells to IL-2-independent growth. Rapamycin, an inhibitor of mTOR kinase, reduced the growth of Tax-transformed CTLL-2 cells. Moreover, the transduction of a constitutively active form of Akt in the CTLL-2 cells also induced IL-2-independent growth. Like CTLL-2/Tax, constitutive phosphorylation of p70S6 kinase was detected in the absence of IL-2 in all of the HTLV-1-infected human T-cell lines. These results suggest that Tax activates the mTOR pathway in T cells, and that this activation plays a crucial role in the growth of HTLV-1-infected T cells when a limited amount of IL-2 is available.     

Cell-free Entry of Human T-Cell Leukemia Virus Type 1 to Mouse Cells

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent for adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. Recently we infected newborn mice by inoculating HTLV-1-producing human cells, and found that T-cells, B-cells and granulocytes were infected in vivo. To understand the mechanism of viral-cell interaction and the pathogenesis of HTLV-1 using the mouse model, it is important to clarify the cellular tropism using a cell-free HTLV-1 transmission system. We employed a highly transmissible cell-free HTLV-1 produced by a feline kidney cell line, c77, and studied the susceptibility of 9 kinds of mouse cell lines, EL4, RLml, CTLL-2, J774.1, DA-1, Ba/F3, WEHI-3, NIH3T3 and Bl, and two kinds of human cell lines, Molt-4 and Hut78. HTLV-1 proviral sequence was found by PCR in all 9 mouse cell lines as well as in 2 human cell lines and viral entry was blocked with sera from an HTLV-1 carrier and an adult T-cell leukemia patient. Unexpectedly, mouse cell lines EL4 and RLml and human cell lines Molt-4 and Hut78 showed similar efficiency for viral entry. These results suggest a wide distribution of HTLV-1 receptor in mouse cells.     

Establishment and characterization of a monocytic cell line which expresses the interleukin-2 receptor

A cell line, SCC-3, was established from the pleural fluid of a patient with non-Hodgkin's lymphoma. Interleukin-2 receptor (IL2-R) was found to be expressed on the cell surface by marker analysis, and morphological, cytochemical, and other marker analyses suggested a monocytic lineage. SCC-3 may be useful for studies on the role of IL2-R in monocytic cells.     

Cell-free entry of human T-cell leukemia virus type 1 to mouse cells.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent for adult T-cell leukemia and HTLV-1-associated myelopathy / tropical spastic paraparesis. Recently we infected newborn mice by inoculating HTLV-1-producing human cells, and found that T-cells, B-cells and granulocytes were infected in vivo. To understand the mechanism of viral-cell interaction and the pathogenesis of HTLV-1 using the mouse model, it is important to clarify the cellular tropism using a cell-free HTLV-1 transmission system. We employed a highly transmissible cell-free HTLV-1 produced by a feline kidney cell line, c77, and studied the susceptibility of 9 kinds of mouse cell lines, EL4, RLm1, CTLL-2, J774.1, DA-1, Ba / F3, WEHI-3, NIH3T3 and B1, and two kinds of human cell lines, Molt-4 and Hut78. HTLV-1 proviral sequence was found by PCR in all 9 mouse cell lines as well as in 2 human cell lines and viral entry was blocked with sera from an HTLV-1 carrier and an adult T-cell leukemia patient. Unexpectedly, mouse cell lines EL4 and RLm1 and human cell lines Molt-4 and Hut78 showed similar efficiency for viral entry. These results suggest a wide distribution of HTLV-1 receptor in mouse cells.     

Induction of human glioma-specific cytotoxic T-lymphocyte lines by autologous tumor stimulation and interleukin 2

Summary Two human glioma-specific cytotoxic T-lymphocyte (G-S-CTL) lines were established by autologous tumor stimulation (ATS) with the aid of lectin free interleukin 2 (IL 2). Coculture of patient's peripheral blood lymphocytes and autologous irradiated glioma cells and subsequent addition of partially purified IL 2 enhanced the tumoricidal activity of the lymphocytes. These CTL lines possessed cross-cytotoxic activity against autologous and allogeneic glioma cells and exhibited low cytotoxic activity against non-glial tumor cells. They did not lyse autologous lymphoblasts. This phenomenon suggested the existence of a common gliomaspecific antigen recognized by the CTL lines.T-cell subset depletion test revealed that the major surface phenotype of G-S-CTL line, responsible for cytotoxic activity was OKT 3 positive, OKT 4 negative and OKT 8 positive.G-S-CTL lines were composed of a low proportion of OKT 8 positive subpopulation after primary ATS and successive propagation with IL 2. The proportion of OKT 8 positive subpopulation was increased by secondary ATS, which enhanced the cytotoxic activity to glioma cells more effectively.