Identification of sample-specific sequences in mammalian cDNA and genomic DNA by the novel ligation-mediated subtraction (Limes).

The representational difference analysis (RDA) and other subtraction techniques are used to enrich sample-specific sequences by elimination of ubiquitous sequences existing in both the sample of interest (tester) and the subtraction partner (driver). While applying the RDA to genomic DNA of cutaneous lymphoma cells in order to identify tumor relevant alterations, we predominantly isolated repetitive sequences and artificial repeat-mediated fusion products of otherwise independent PCR fragments (PCR hybrids). Since these products severely interfered with the isolation of tester-specific fragments, we developed a considerably more robust and efficient approach, termed ligation-mediated subtraction (Limes). In first applications of Limes, genomic sequences and/or transcripts of genes involved in the regulation of transcription, such as transforming growth factor beta stimulated clone 22 related gene (TSC-22R), cell death and cytokine production (caspase-1) or antigen presentation (HLA class II sequences), were found to be completely absent in a cutaneous lymphoma line. On the assumption that mutations in tumor-relevant genes can affect their transcription pattern, a protocol was developed and successfully applied that allows the identification of such sequences. Due to these results, Limes may substitute/supplement other subtraction/comparison techniques such as RDA or DNA microarray techniques in a variety of different research fields.

Inhibition of constitutively activated Stat3 correlates with altered Bcl-2/Bax expression and induction of apoptosis in mycosis fungoides tumor cells.

The Jak/Stat signaling pathway transmits signals from many cytokine and growth factor receptors to target genes in the nucleus. Constitutive activation of Stat3 has recently been observed in many tumor cells and dysregulation of the Stat signaling pathway has been proposed to be implicated in malignant transformation. In a previous study, we found constitutively tyrosine phosphorylated Stat3 in mycosis fungoides tumor cells. Here, we show that the Jak kinase inhibitor, Ag490, inhibits the constitutive binding of Stat3 to an oligonucleotide representing the Stat-binding sequence from the ICAM promotor. The decreased ability of Stat3 to bind DNA precedes dynamic alterations in the expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax proteins (decreased Bcl-2 expression and increased Bax expression) and induction of apoptosis. Thus, our data suggest that the involvement of Stat3 in oncogenic transformation could be mediated through regulation of survival signals.

Constitutive STAT3-activation in Sezary syndrome: tyrphostin AG490 inhibits STAT3-activation, interleukin-2 receptor expression and growth of leukemic Sezary cells.

Interleukin-2 (IL-2) is a growth factor which upon binding to high-affinity receptors (IL-2Ralphabetagamma) triggers mitogenesis in T cells. IL-2Ralpha expression is restricted to T cells which have recently encountered antigen, and in healthy individuals the majority (>95%) of peripheral T cells are IL-2Ralpha negative. An aberrant expression of IL-2Ralpha has recently been described in cutaneous T-cell lymphoma (CTCL). Here, we study the regulation of IL-2Ralpha expression and STATs in a tumor cell line obtained from peripheral blood from a patient with Sezary syndrome (SS), a leukemic variant of CTCL. We show that (1) STAT3 (a transcription factor known to regulate IL-2Ralpha transcription) is constitutively tyrosine-phosphorylated in SS tumor cells, but not in non-malignant T cells; (2) STAT3 binds constitutively to a STAT-binding sequence in the promotor of the IL-2Ralpha gene; (3) the Janus kinase inhibitor, tyrphostine AG490, inhibits STAT3 activation, STAT3 DNA binding, and IL-2Ralpha mRNA and protein expression in parallel; and (4) tyrphostine AG490 inhibits IL-2 driven mitogenesis and triggers apoptosis in SS tumor cells. In conclusion, we provide the first example of a constitutive STAT3 activation in SS tumor cells. Moreover, our findings suggest that STAT3 activation might play an important role in the constitutive IL-2Ralpha expression, survival, and growth of malignant SS cells.

Plasminogen activating enzyme in cultured glioblastoma cells. An immunofluorescence study with monoclonal antibody.

A monoclonal antibody against a 52,000 dalton human plasminogen activating enzyme (HPA52) was used for immunofluorescence staining of cultured glioblastoma cells. The fluorescence was located in the cytoplasm of the cells. A pronounced variation in the staining intensity was observed between the individual cells. The specificity of the fluorescent stain was supported by the findings that 1) no staining was obtained with a monoclonal antibody of the same subclass, but with irrelevant specificity (anti-2,4,6-trinitrophenyl); 2) adsorption with HPA52 purified to homogeneity removed the ability of anti-HPA52 to mediate staining; 3) the glioblastoma cells contained HPA52, as measured by enzymatic assay, while melanoma cells that were not stained did not contain HPA52 activity; 4) dexamethasone reduced both the enzymatically determined HPA52 content and the immunofluorescence in parallel, while progesterone affected none of these parameters; 5) we have previously found that culture fluid conditioned by the glioblastoma cells apart from HPA52 does not contain detectable amounts of any protein that binds to anti-HPA52. Several advantages of immunohistochemical detection of plasminogen activators compared with enzyme histochemical methods are discussed, among these that the immunohistochemical method distinguishes between plasminogen activators of different types.

Solid-phase iodothyronine-5'-deiodinase (5'-D) assays applied in production of monoclonal antibodies against 5'-D.

Characterization of iodothyronine-deiodinating enzymes has been difficult due to loss of enzyme activity during purification. To obtain a new tool for studying these enzymes we investigated the possibility of developing monoclonal antibodies (MAbs) against iodothyronine-5'-deiodinase (5'-D). Two specific and sensitive solid-phase microassays were developed for screening hybridoma supernatants for the presence of antibodies inhibiting rat kidney 5'-D and antibodies binding to but not inhibiting the enzyme. BALB/c mice were immunized with a 3-((3-cholamidopropyl)-dimethylammonio)-1-propanesulphonate (CHAPS)-solubilized 5'-D-rich membrane preparation from rat kidney cortical tissue. Spleen cells were fused with NSI-Ag 4/l mouse myeloma cells by means of polyethylene glycol. Two hybridoma cell lines (AF5 and BE8) secreting MAbs specifically binding to without inhibiting 5'-D were produced. The AF5 antibody was of the IgG2a subclass and the BE8 antibody of the IgG2b subclass. Binding of one of the antibodies to the enzyme inhibited binding of the other in both an enzyme-linked immunosorbent assay (ELISA) and a specific enzyme-binding assay. CHAPS-solubilized kidney microsomal fraction was chromatographed on a Sepharose 6B column. Elution profiles of 5'-D activity and MAb-binding antigens, as measured by ELISA with both AF5 and BE8, were identical. Monoclonal antibodies should be valuable probes in the further elucidation of the nature of the iodothyronine-deiodinating activity in various tissues.

Appearance of isochromosome 18q can be associated with in vitro immortalization of human T lymphocytes.

T lymphocytes cultured from a skin biopsy specimen of a patient with atopic dermatitis developed isochromosome 18q concomitant to escape from replicative senescence. Furthermore, two T-cell lines established from patients with cutaneous T-cell lymphoma also developed isochromosome 18q during continuous growth. The results indicate that a pathway leading to immortalization of human T lymphocytes could involve genes located at chromosome 18.

Microdissection and reverse painting in a melanoma cell line: a detailed description of structurally abnormal chromosomes.

Seven structurally abnormal chromosomes from a newly established melanoma cell line were microdissected. The DNA from these chromosomes was DOP amplified and labeled with Biotin or Digoxigenin for FISH analysis. The complex nature of these markers is described.

Common clonal chromosome aberrations in cytokine-dependent continuous human T-lymphocyte cell lines.

Antigen-mediated T-cell proliferation is a transient phenomenon. Like other somatic cells, T lymphocytes generally show replicative senescence in vitro. However, we here show that cytokine-dependent continuous (immortal) T-cell lines can be established from skin biopsy specimens of inflammatory skin diseases. Continuous growth can be obtained by culturing T cells in medium supplemented with interleukin-2 and interleukin-4, but without antigen or antigen-presenting cells added. Loss of the T-cell antigen receptor complex is observed in some of the continuous T-cell lines. Most T-cell lines develop clonal chromosome aberrations during continuous growth. Aberrations for chromosomes 1, 2, 8, 16, and 18 are most commonly observed.

Aneuploid malignant T cells from a patient with Sézary syndrome can be visualized by in situ hybridization.

BACKGROUND AND DESIGN: Enumeration of malignant cells in Sézary syndrome often relies on the identification of the Sézary cell nucleus. This morphologic method is, however, nonspecific and unreliable in the enumeration of the proportion of malignant lymphocytes in peripheral blood of patients with Sézary syndrome. Malignant lymphocytes of patients with mycosis fungoides and Sézary syndrome are often characterized by multiple chromosome aberrations. Herein, we demonstrate that fluorescent in situ hybridization can visualize and accurately enumerate malignant aneuploid mononuclear cells in a patient with Sézary syndrome. RESULTS: Fluorescent in situ hybridization demonstrated that 90% of the mononuclear cells in the patient with Sézary syndrome showed numerical aberrations for both chromosome 7 and X, a figure confirmed by flow cytometry. CONCLUSION: Fluorescent in situ hybridization may be a valuable tool to visualize and enumerate aneuploid tumor cells in patients with cutaneous T-cell lymphoma.

Lck is involved in interleukin-2 induced proliferation but not cell survival in human T cells through a MAP kinase-independent pathway.

The role of Lck in IL-2-induced proliferation and cell survival is still controversial. Here, we show that the Src family kinase inhibitor, PP1, reduced the IL-2-induced proliferation of human T cells significantly without inhibiting the anti-apoptotic effect of IL-2. As Lck is the only Src family kinase activated upon IL-2 stimulation in T cells, this indicates that Lck is involved in IL-2-induced proliferation but not survival. IL-2-induced MAP kinase activation was only slightly inhibited by PP1, suggesting that Lck is not essential for IL-2-induced MAP kinase activation in human T cells. We found that an IL-2-sensitive, human mycosis fungoides-derived tumor T cell line is Lck negative, and that the IL-2-induced MAP kinase activation is comparable to non-cancerous T cells, although a little delayed in kinetics. An Lck expressing clone was established by transfecting Lck into mycosis fungoides tumor T cells, but Lck had no influence on the delayed kinetics of MAP kinase activation, indicating that Lck is not essential for MAP kinase activation in mycosis fungoides tumor T cells or in non-cancerous T cells. Taken together, this indicates that Lck is involved in IL-2-induced proliferation, but not cell survival, through a pathway not involving MAP kinase.

Genotraumatic T cells and cutaneous T-cell lymphoma. A causal relationship?

Mycosis fungoides, or cutaneous T-cell lymphoma (CTCL), is a T-cell mediated chronic inflammatory skin disease, which can occasionally progress with a variable time course to a fatal lymphoma or to a leukaemic form called Sézary's syndrome. Extensive research into CTCL has not yet elucidated the primary pathophysiological mechanisms. Immunohistological studies are so far less helpful than expected in establishing early diagnosis and prognosis of the disease. The proposition that an exogenous virus is the cause of CTCL has not been substantiated. Karyotypic analysis of lymphocytes from the skin and blood of patients with CTCL have shown the existence of several genetically aberrant T-cell clones in the same patient. These changes are discussed as potential primary events for the development of CTCL. The hypothesis is put forward that the development of genotraumatic T lymphocytes is involved in the progression of the disease.

Human T lymphocytes and T-cell lines as target cells for lymphocyte chemotaxis.

We have observed that freshly isolated T lymphocytes from healthy donors give a chemotactic response to complement C5a in 26 of 55 individuals and to epidermal lymphocyte chemotactic factor in 15 of 23 donors using 51chromium-labelled lymphocytes in a double-filter Boyden chamber system. The reason for a lack of demonstrable chemotaxis among some cell populations is unknown, but it makes donor selection important when studying lymphocyte chemotaxis. In order to obtain a standardized screening assay for T lymphocyte chemotactic activity, we investigated a number of T-cell lines or T-cell-related cell lines such as HuT78, Jurkat, MOLT4, K562 and 1301. We observed that HuT78, K562 and Jurkat showed chemotactic responses to a variety of mediators, whereas 1301 showed chemotaxis only towards C5a, and MOLT4 was completely negative. The HuT78 cell line, which is derived from a patient with Sézary's syndrome, exhibited the highest chemotactic capacity similar to freshly isolated T lymphocytes. The only difference was its chemotactic response towards stimulation with recombinant interleukin-1 alpha and beta, which did not induce chemotaxis in human peripheral blood T lymphocytes in the Boyden chamber assay. We conclude that HuT78 can be used in screening various inflammatory mediators for their potential T lymphocyte chemotactic properties.

A majority of proliferating T cells in cutaneous malignant T cell lymphomas may lack the high affinity IL-2 receptor (CD25).

IL-2 is a major growth factor for all T-cell subsets acting via a specific membrane receptor. To investigate its role in the pathogenesis of cutaneous T-cell lymphomas (CTCLs), we analysed the expression of high-affinity IL-2 receptors (IL-2Rs) on proliferating cells in these disorders. We showed by immunohistochemical double staining that most cycling cells do not express high-affinity IL-2Rs. Four T-cell lines were established from patients with CTCLs. Two lines required both IL-2 and IL-4 for growth, one line required IL-2 and one line IL-4 alone. The last of these lacked expression of the IL-2R alpha-chain. Thus, IL-2 may not be the only, or the most important, growth stimulus in CTCLs and reactive skin infiltrates. T helper cells, which dominated the infiltrate, might represent TH2 cells.

Natural and concanavalin A-induced cytotoxic activity towards continuously growing B lymphocytes derived from patients with cutaneous T-cell lymphoma.

Continuously growing T- and B-cell lines were derived from peripheral blood, affected skin, and lymph nodes of patients with mycosis fungoides (MF) and Sézary syndrome (SS). Two lymphoblastoid cell lines (MF-13 and SS-2) were Epstein-Barr virus (EBV)-transformed B cells evaluated by surface immunoglobulin, lack of E-rosette formation, positive EBV nuclear antibody test, and secretion of IgM antibody in a plaque-forming cell assay. Analysis of the natural-killer-cell activity using peripheral blood lymphocytes from patients with MF and healthy control persons towards MF-13 and SS-2 target cells suggested resistance to lysis even in tests supplemented with 1,000 IU/ml human gamma-interferon. However, the cell lines were not per se completely resistant to lysis because lymphocytes from control persons showed significant cytotoxicity in an 18-h assay supplemented with 2 micrograms/ml concanavalin A.

In vitro genetically aberrant T-cell clones with continuous growth are associated with atopic dermatitis.

Atopic dermatitis is a disease with a genetic predisposition affecting the immune system, with T lymphocytes participating in the immune dysregulation. Most in vitro T lymphocyte studies of atopic dermatitis have focused on antigen-specific T-cell clones. However, antigen-non-specific regulatory T lymphocytes may also take part in the pathway leading to antigen-specific clonal T-lymphocyte proliferation. T lymphocytes from skin biopsy specimens from three patients with severe atopic dermatitis were cultured in the presence of IL-2 and IL-4, but without antigen added. Initially, proliferation was oligo- or polyclonal, but in all cases overgrowth by T cells with clonal chromosomal aberrations was subsequently observed. These abnormal T-cell clones demonstrated continuous growth and complete or partial phenotypic loss of the T-cell antigen receptor complex. In summary, these findings suggest that a subset of aberrant skin-homing T lymphocytes is associated with atopic dermatitis.

Phenotypic determination of T-lymphocytes responding to chemotactic stimulation from fMLP, IL-8, human IL-10, and epidermal lymphocyte chemotactic factor.

Human T lymphocytes were collected after they had migrated towards N-formyl-methionyl-leukylphenylalanine (fMLP), rIL-8, human IL-10 (hIL-10), and epidermal lymphocyte chemotactic factor (ELCF). They were stained for determination of their phenotype by FACS analysis using anti-CD4, -CD8, -CD18, -CD45R0 and OPD4 antibodies. Human IL-10 increased the percentage of CD8+ T lymphocytes in the migrating cell population by 152% compared with cells migrating towards the medium and decreased the number of CD4+ T lymphocytes by 79%. ELCF increased the number of CD4+ T lymphocytes by 18%, and the number of CD45R0+ T lymphocytes by 52%, while the number of CD8+ T lymphocytes was decreased by 20%. rIL-8 increased the number of CD4+ T lymphocytes and decreased the CD8+ T lymphocytes. The distribution of the different subpopulations of T lymphocytes was not changed significantly by fMLP. The observed changes in the phenotypes did not occur when incubating T lymphocytes with the chemotaxins. Our observations demonstrate that individual chemotactic factors will attract specific subsets of T lymphocytes. They may help to explain the predominance of memory T lymphocytes (CD4R0+, CD4+) in allergic contact dermatitis and certain other skin diseases. They also confirm the results of a recent study, that showed hIL-10 to be selectively chemotactic for CD8+ T lymphocytes.

A continuous T-cell line from a patient with Sézary syndrome.

A continuous cell line, Se-Ax, from a patient with Sézary syndrome has been established. The Se-Ax cell line is IL-2 dependent, requires human serum for permanent growth, and has the following phenotype: CD1-, CD2+, CD3+, CD4-, CD5-, CD8-, CD20-, CD25+; it expresses the T9, T10, and HLA-DR antigens. This cell line reveals multiple chromosome aberrations as seen in the most abundant abnormal clone in peripheral blood. Therefore, it is not unlikely that it derives from tumor cells. A putative cytotoxic cell line derived from the same patient has only weak killer-cell activity against the autologous permanent cell line: this CD8+ cytotoxic cell line has a 14q+ chromosomal marker. The fact that the patient demonstrated no natural killer-cell or activated killer-cell activity against the Se-Ax cell line may in part explain the successful establishment of the continuous cell line from bulk culture.

C-type particles are inducible in Se-Ax, a continuous T-cell line from a patient with Sézary's syndrome.

Se-Ax is a continuous mature T-cell line that we have established from a patient with Sézary's syndrome. An important finding was that the Se-Ax cell line required human serum for initial growth. Here we show that transfer of the permanent cell line to a medium deficient of human serum induces production of C-type retroviral-like particles with a unique morphology. Ultrastructurally, these particles are 120 nm in diameter with hexagonal shape, and show a small, centrally located round core of 30 nm. They are observed only extra-cellularly; typical budding, however, is not found. Both by serological testing and molecular analysis we substantiate the morphological conclusion that these particles do not represent common human or animal retroviruses. The inducibility of the particles may be a hint as to a possible endogenous origin.

Cytogenetic findings in cell lines from cutaneous T-cell lymphoma.

Cytogenetic analysis of some patients with CTCL demonstrates the presence of more than one cytogenetically aberrant clone in a given patient. These findings lead us to suggest that CTCL is defined by a family of "genotraumatic" T cells. A genotraumatic T cell, unlike a normal T lymphocyte, is defined by its ability to develop clonal, cytogenetically visible chromosome aberrations. Based on this hypothesis, an investigation was performed in detail of cell lines from the plaques and blood of a patient with MF. Several genotraumatic T cells could be demonstrated. Of particular interest was the establishment of two continuous T-cell lines from a single plaque. Both genotraumatic T-cell lines were genetically unstable, and multiple and complex chromosome aberrations could be demonstrated in both cell lines, suggesting that two potentially malignant T-cell clones exist in a single plaque. It is proposed that CTCL is defined by a family of genotraumatic T cells and is thus, in principle, oligoclonal or polyclonal. All genotraumatic T cells may be considered cancer prone because of their ability to develop clonal chromosomal aberrations. A genotraumatic T cell is per se not malignant, but owing to its genetic instability, it may develop into a tumor cell. This could explain how an apparent benign disorder, CTCL, occasionally may progress into malignant lymphoma.

Balanced terminal chromosome translocations develop in EBV-derived, but non-immortal cell lines from patients with mycosis fungoides.

Five lymphoblastoid cell lines established from patients with mycosis fungoides were followed during long-term culture. They all expressed the Epstein-Barr virus nuclear antigen. Although the established B-cell lines initially showed a normal karyotype, metaphases with balanced terminal translocations gradually occurred. These chromosome abnormalities occurred simultaneously with decreased cell proliferation leading in all cases to termination of the cell lines. This contrasts the usual finding that Epstein-Barr virus established B-lymphoblastoid cell lines are immortal.