Michael Notter

Cellular effects of CPT-11 on colon carcinoma cells: Dependence on p53 and hMLH1 status

Irinotecan (CPT‐11), a recently introduced component of a standard chemotherapy for colorectal cancer, induces in colon cancer cell lines in vitro cell cycle arrest and apoptosis. Since sporadic colon carcinomas exhibit in 50–60% mutations in the p53 gene and in 10–15% an MSI phenotype due in the great majority of the cases to hMLH1 inactivation, we investigated how these lesions influence the cellular effects of CPT‐11 by using colorectal carcinoma cell line HCT116 (which has the genotype p53+/+,hMLH1−) and 2 derivative cell lines with the genotypes p53+/+,hMLH1+ and p53−/−,hMLH1−. CPT‐11 treatment induced G2/M arrest in all 3 cell lines within 48 hr. In the p53+/+,hMLH1+ cell line, G2/M arrest was maintained for at least 12 days. There was little concomitant apoptosis, but this was enhanced when the hMLH1 protein was absent. This enhanced apoptosis was accompanied by a shorter duration of the G2/M arrest than in the hMLH1+ cell line. Partial abrogation of G2/M arrest by caffeine enhanced apoptosis in both hMLH1+ and hMLH1− cells. By contrast, in the p53−/− cell line, the G2/M arrest was terminated within 4 days. Termination of the G2/M arrest was accompanied by a high level of apoptosis detectable through poly(ADP‐ribose)polymerase (PARP) cleavage, DNA fragmentation and by the appearance of cells with a DNA content <2N. The triggering of G2/M arrest was accompanied in the 3 cell lines by a transient phosphorylation of cdc‐2, while the maintenance of the arrest in the p53+/+ cell lines was accompanied by the overexpression of p53 and p21 proteins and, consequently, by the inhibition of cdc‐2 kinase activity. These data indicate that: (i) CPT‐11 induces long‐term arrest in p53+/+ cells and a short‐term arrest followed by apoptosis in p53−/− cells; (ii) triggering of the arrest is p53 independent and is associated with a brief increase of phosphorylation of cdc‐2, while the p53‐dependent maintenance of G2/M arrest is associated with the inhibition of cdc‐2 kinase activity by p21; and (iii) lack of hMLH1 protein enhances CPT‐11‐induced apoptosis. These results may be useful for designing rational therapies dependent on the p53 and mismatch‐repair status in the tumor.

Incidence and Prognostic Significance of Immunophenotypic Subgroups in Childhood Acute Lymphoblastic Leukemia: Experience of the BFM Study 86

Due to the increasing availability of monoclonal antibodies (MAbs) recognizing lymphoid-, myeloid-, and progenitor-cell-associated antigens, immunophenotyping has greatly influenced studies on the biologic features of normal and leukemic hematopoietic progenitor cells (Greaves 1986). It has become possible to demonstrate the practical value of these data for the precise diagnosis and definition of clinically relevant immunophenotypic subsets of acute lymphoblastic leukemia (ALL) (Janossy et al. 1989). More recently, immunophenotyping has been supplemented by cytogenetic and molecular-genetic analyses in order to better characterize the biologic heterogeneity of ALL and to elucidate the mechanisms of lymphoid cell transformation and aberrant regulation of leukemic cell growth (Look 1988; Bain and Catovsky 1990; Pui et al. 1990c; van Dongen and Wolvers-Tettero 1991).

The Elongated First Fibronectin Type III Domain of Collagen XIV Is an Inducer of Quiescence and Differentiation in Fibroblasts and Preadipocytes

Collagen XIV (CXIV) is a fibril-associated collagen that is mainly expressed in well differentiated tissues and in late embryonic development. Because CXIV is almost absent in proliferating and/or dedifferentiated tissues, a functional role in maintaining cell differentiation is suspected. We demonstrate antiproliferative, quiescence- and differentiation-inducing effects of human CXIV and its recombinant fragments on mesenchymal cells. In primary human fibroblasts, in mouse 3T3 fibroblasts and in 3T3-L1 preadipocytes, CXIV reduced de novo DNA synthesis by 75%, whereas cell numbers and viability remained unaltered. Cells showed no signs of apoptosis, and maximal proliferation was restored when serum was supplemented, thus indicating that CXIV induced reversible cellular quiescence. Exposure of fibroblasts to CXIV in vitro led to cellular bundles and clusters. CXIV also triggered trans-differentiation of 3T3-L1 preadipocytes into adipocytes, as could be shown by lipid accumulation and by expression of the glucose transporter Glut4. These effects were also observed with the amino-terminal recombinant fragment Gln29-Pro154 that harbors the first fibronectin type III domain and a 39-amino-acid extension, whereas no activity was found for all other recombinant CXIV fragments. Based on these finding the development of small molecular analogs that modulate fibroblast cell growth and differentiation, e.g. in wound healing and fibrosis, seems feasible.

Expression of interleukin 15 in primary adult acute lymphoblastic leukemia

Interleukin‐15 (IL‐15) has been associated with the growth, survival and biological behavior of leukemic cells and response to therapy. We determined the expression of IL‐15 in lymphoblasts and evaluated its potential impact on the outcome in adult acute lymphoblastic leukemia (ALL).

CD82 (KAI1), a member of the tetraspan family, is expressed on early haemopoietic progenitor cells and up‐regulated in distinct human leukaemias

CD82 (KAI1) is a member of the tetraspan transmembrane protein family which has been cloned from lymphoblastoid variant cell lines. However, a role for CD82 in early normal and malignant haemopoiesis has not yet been characterized. We studied the CD82 expression in 33 normal donor samples and 98 leukaemias by fluorescence activated cell sorting (FACS) and reverse transcriptase polymerase chain reaction (RT‐PCR). We demonstrated that CD82 was moderately expressed in the vast majority of normal granulocytes and monocytes. In contrast, only about one third of the peripheral blood lymphocytes were weakly CD82 positive (CD82+). Interestingly, judgement of the CD82 transcription and expression in various leukaemias revealed that CD82 was overexpressed in chronic myeloid leukaemia (CML) patients in accelerated or blastic phase (CML‐AP/BP) as well as in acute myeloid leukaemia (AML) and chronic lymphocytic leukaemia (CLL) patients. Analysis of AML patients with CD34+/CD82+ blasts prompted us to expand our studies on haemopoietic CD34+ progenitor cells. Intriguingly, 84–95% of the CD34+ cells isolated from healthy bone marrow, cord blood or peripheral blood were highly CD82+. CD82 was abundantly expressed on primitive as well as on committed haemopoietic progenitor cells. After in vitro induction of myeloid differentiation in CD34+ peripheral blood progenitor cells (PBPC), the expression of CD82 decreased to levels similar to those found on peripheral blood granulocytes. These observations suggest for the first time a role for CD82 in normal and malignant haemopoiesis.

Efficacy and pharmacologic data of second-generation tyrosine kinase inhibitor nilotinib in BCR-ABL-positive leukemia patients with central nervous system relapse after allogeneic stem cell transplantation.

Central nervous system (CNS) involvement is a severe complication of BCR-ABL-positive leukemia after allogenic stem cell transplantation (alloSCT) associated with fatal outcome. Although second-generation tyrosine-kinase inhibitors (TKI) such as nilotinib have shown activity in systemic BCR-ABL+ disease, little data exists on their penetration and efficacy within the CNS. Four patients (3 male, 1 female; age 15–49) with meningeal relapse after alloSCT and subsequent treatment with nilotinib were identified. A total of 17 cerebrospinal fluid (csf) and serum samples were assessed for nilotinib concentration and patient outcome was recorded. Nilotinib concentrations showed a low median csf/plasma ratio of 0.53% (range 0.23–1.5%), yet pronounced clinical efficacy was observed with long-lasting responses (>1 year) in three patients. Comparison with historical data showed a trend towards superior efficacy of nilotinib versus imatinib. Despite poor csf penetration, nilotinib showed significant clinical activity in CNS relapse of BCR-ABL+ leukemias. As nilotinib has a high protein-binding affinity, the low-protein concentration in csf could translate into a relatively higher amount of free and therefore active nilotinib in csf as compared to blood, possibly explaining the observed efficacy. Thus, treatment with a 2nd generation TKI warrants further investigation and should be considered in cases of CNS relapse of BCR-ABL-positive leukemia after alloSCT.

CS‐1, a novel c‐kithi+ acute myeloid leukemia cell line with dendritic cell differentiation capacity and absent immunogenicity

Complex cytogenetic abnormalities confer dismal prognoses in myeloid malignancies. Even bone marrow transplantation from siblings or matched unrelated donors offer minimal chances for cure, suggesting that these cases are not only refractory to chemotherapy but also resist the graft‐vs.‐leukemia effect. We herein describe the first permanent, factor‐independent c‐kithi+ cell line CS‐1 derived from an unrelated donor stem cell transplanted patient with relapsed acute myeloid leukemia (AML)‐M5a of high‐risk karyotype [monosomy 7, t(2;11)(q31;p13), t(10;12)(q24;q24)]. Having the same karyotype, CS‐1 exhibits an autonomous growth pattern and responds to stem cell factor (SCF). CS‐1 did not induce T cell activation in mixed‐lymphocyte‐tumor‐cultures (MLTCs) and, when used as third party stimulators, decreased T cell proliferation in mixed‐lymphocyte reactions (MLRs). Cytokines added exogenously or secreted from bystander T cells caused CS‐1 to differentiate into dendritic cells (DCs). CS‐1‐derived DCs, in contrast to DCs originating from non‐malignant CD34+ progenitor cells, had virtually no T cell stimulatory effect, indicating that CS‐1 is both immunosuppressive and poorly immunogenic. These properties may partially be due to the detected downregulation of costimulatory molecules and appear to involve a soluble factor. CS‐1 cells injected subcutaneously (s.c.) to non‐obese diabetes/severe combined immunodeficient (NOD/SCID) mice produced solid tumors, disseminating into bone marrow and spleen. The data show that transforming AML blasts with high‐risk karyotype into DCs is insufficient to restore their immunogenicity and that the CS‐1 cell line is useful to identify tumor‐related immunosuppressive mechanisms in vitro and in vivo.

Preclinical Studies of T-Cell-Mediated Immune Responses Against Autologous Tumor Cells in Patients with Acute Leukemia

Risk-adapted therapy in patients with acute leukemia has significantly improved prognosis. As a result of relapsing disease and increasing resistance to chemotherapy, however, long-term survival in nontransplanted patients does not exceed 30% (Buchner et al. 1985). Clearly, new therapeutic approaches are needed.

Zwei ungewöhnliche kutane T-Zell-Lymphome mit extrakutaner Beteiligung

ZusammenfassungWir stellen zwei Patienten mit ungewöhnlichen Hautveränderungen als erstem klinischen Symptom eines kutanen T-Zell-Lymphoms mit hautüberschreitender Organmanifestation vor. Bei den Hautveränderungen handelte es sich (a) um panzerartige, ödematöse Infiltrationen von Bauch und Beinen sowie um gruppiert stehende, erythematöse, zentral genabelte Papeln in den großen Körperfalten und (b) um großflächige Erytheme, Teleangiektasien, Pigmentverschiebungen und blitzfigurenartige Narben im Sinne eines ungewöhnlichen Poikiloderma vasculare atrophicans Jacobi, auf dem sich im Verlauf der Erkrankung Papeln und Tumorknoten entwikkelten. Unsere erstere Patientin hatte zudem eine Leukozytose und eine Vergrößerung der hautnahen Lymphknoen; Histologie, Immunohistologie und Immunphänotypisierung zeigten, daß sie an einem schwer klassifizierbaren CD8+, α,β+, CD10+T-Zell Lymphom erkrankt war. Bei dem zweiten Patienten wurde ebenfalls ein schwer klassifizierbares T-Zell-Lymphom diagnostiziert, das mit einer monoklonalen Vermehrung von T-Zell-Rezeptor Vγ9,δ+großen granulären T-Lymphozyten einherging und sich im Verlauf der Erkrankung in eine akute prä-T-lymphatische Leukämie entwikkelte.SummaryIn this paper we present two patients with T-cell lymphomas, who presented with unique skin lesions, but later developed extracutaneous involvement. The first patient showed marked edematous infiltration of the trunk and legs as well as grouped, umbilicated red-to-blue papules limited to the body folds. The second patient had lesions reminiscent of poikiloderma vasculare atrophicans, with erythema, teleangiectasia, hypo- and hyperpigmentation, and bizarre scarring, followed by development of papules and nodules. The first patient also had marked leukocytosis and lymph node enlargement. Histology, immunohistology, and immunophenotyping revealed an unclassifiable CD8+, α,β+, CD10+ cutaneous T-cell lymphoma with leukemic involvement. Unclassifiable cutaneous T-cell lymphoma was also diagnosted in the second patient: it was associated with a monoclonal proliferation of T-cell receptor Vγ9,δ+ large granular T lymphocytes and finally developed into frank, acute pre-T lymphatic leukemia.