Colette Adida

INDUCTION OF APOPTOSIS AND INHIBITION OF CELL PROLIFERATION BY SURVIVIN GENE TARGETING

Survivin is a new IAP apoptosis inhibitor expressed during development and in human cancer in vivo. The coding strand of the survivin gene was extensively complementary to that of effector cell protease receptor-1 (EPR-1), prompting the present investigation on the origin and functional relationship of these two transcripts. Southern blots of genomic DNA were consistent with the presence of multiple, evolutionarily conserved, EPR-1/Survivin-related genes. By pulsed field gel electrophoresis and single- and two-color fluorescence in situ hybridization, these were contained within a contiguous physical interval of 75–130 kilobases (kb) on chromosome 17q25. In Northern blots, a single strand-specific probe identified a 1.3-kb EPR-1 mRNA broadly distributed in normal adult and fetal tissues, structurally distinct from the 1.9-kb Survivin transcript expressed in transformed cell lines. Transient co-transfection of an EPR-1 cDNA potentially acting as a Survivin antisense with a lacZ reporter plasmid resulted in loss of viability of HeLa cells. In contrast, co-transfection of an antisense cDNA of intercellular adhesion molecule-1 or a sense-oriented Survivin cDNA was without effect. In stably transfected HeLa cells, ZnSO4 induction of an EPR-1 mRNA under the control of a metallothionein promoter suppressed the expression of endogenous survivin. This resulted in (i) increased apoptosis as detected by analysis of DNA content and in situ internucleosomal DNA fragmentation and (ii) inhibition of cell proliferation as compared with induced vector control transfectants. These findings suggest the existence of a potential EPR-1/survivin gene cluster and identify survivin as a new target for disrupting cell viability pathways in cancer.

Anti-apoptosis gene, survivin, and prognosis of neuroblastoma

Neuroblastoma may be aggressive with a dismal outcome or regress spontaneously. Prognostic factors including age, stage, and ploidy, N-myc amplification, and histology are necessary to direct treatment. Gene products controlling apoptosis may modulate neuroblastoma’s variable course. Although expression of anti-apoptotic bcl-2 has been linked to a more aggressive disease, and inversely correlated with morphological features of apoptosis, it is unclear whether inhibition or apoptosis is a predictive factor in neuroblastoma or whether it reflects the degree of cell differentiation. An apoptosis inhibitor, survivin, has recently been identified. Unlike other members of the bcl-2 and IAP gene families, survivin is selectively expressed in all the most common human cancers but not in normal adult tissues. We determined the expression of survivin in neuroblastoma and its relation to disease progression. We studied 72 cases of neuroblastoma for expression of survivin by immunohistochemistry and immunoblotting and correlated our results with clinical stage and histological classification (Shimada). Specific staining for survivin was observed in 34 cases (47%) with variable numbers of positive cells (5–90%). Cases were scored positive when more than 5% of cells reacted with the anti-survivin antibody. When stratified for favourable/unfavourable histology, expression of survivin significantly (p=0·001) segregated with the unfavourable group (17/23, 73·9%), whereas the absence of survivin coincided with favourable histology (32/49, 65%). 14 of 17 cases (82%) with favourable histology which expressed survivin also stained positive for bcl-2 and AChE activity, did not change with age, whereas mean cortical CBF values measured with IMP-SPECT significantly decreased (figure). Since K1 equals the product of extraction fraction and CBF, K1 and CBF should correlate. The estimated mean cortical K1 values, simultaneously obtained with the calculation of k3, also decrease with age in the same fashion as CBF (data not shown, Y=20·0020X+0·62, r=0·356), indicating validity of the kinetic model. Cholinergic enzyme activity levels have been reported to decrease or remain unchanged with age in studies of necropsy brain. The biochemical studies in normal human ageing with necropsy brain specimens have limited value since not only lag time from death to sampling but also duration and condition preceding death of the patients may have great influences on the results. Therefore, a problem in research on neurochemistry of human ageing is the lack of fresh non-diseased brain tissue for analysis. In vivo noninvasive measurement of enzyme activity enables us to circumvent these drawbacks and gives us useful information. The results of this study show that AChE activity of the cerebral cortex does not change with age whereas CBF decreases, suggesting a preserved ascending cholinergic system to the cerebral cortex from the basal forebrain in normal ageing. We conclude that biochemical processes in the cholinergic system underlying ageing and dementia are different.

Control of Apoptosis during Angiogenesis by Survivin Expression in Endothelial Cells

Mechanisms controlling endothelial cell survival during angiogenesis were investigated. Stimulation of quiescent endothelial cells with mitogens, including vascular endothelial growth factor and basic fibroblast growth factor, induced up to approximately 16-fold up-regulation of the cell cycle-regulated apoptosis inhibitor survivin. Mitogen stimulation rapidly increased survivin RNA expression in endothelial cells, which peaked after 6 to 10 hours in culture and decreased by 24 hours. Inflammatory cytokines, tumor necrosis factor alpha, and interleukin-1 did not induce survivin expression in endothelial cells. Formation of three-dimensional vascular tubes in vitro was associated with strong induction of survivin in endothelial cells, as compared with two-dimensional cultures. By immunohistochemistry, survivin was minimally expressed in endothelium of nonproliferating capillaries of normal skin, whereas it became massively up-regulated in newly formed blood vessels of granulation tissue in vivo. Recombinant expression of green fluorescent protein survivin in endothelial cells reduced caspase-3 activity and counteracted apoptosis induced by tumor necrosis factor alpha/cycloheximide. These findings identify survivin as a novel growth factor-inducible protective gene expressed by endothelial cells during angiogenesis. Therapeutic manipulation of survivin expression and function in endothelium may influence compensatory or pathological (tumor) angiogenesis.

Expression and prognostic significance of survivin in de novo acute myeloid leukaemia

Survivin is an inhibitor of apoptosis (programmed cell death) overexpressed in various human cancers, but undetectable in normal differentiated tissues. A potential distribution and prognostic significance of survivin in patients with de novo acute myeloid leukaemia (AML) was investigated. By immunofluoresence of bone‐marrow specimens and peripheral blood mononuclear cells, survivin was detected in 75 out of 125 interpretable AML cases (60%), with reactivity in 50–90% of AML cells. Survivin expression correlated with a lower white blood cell count (WBC) (P = 0·008 by the Mann–Whitney test) and was associated, in the 55 cases of FAB M0/M1/M2, with leukaemic granulocytic maturation (one out of five M/L0, 11 out of 22 M/L1 and 23 out of 28M/L2; P = 0·007 by the Fisher test). In 69 patients treated with the Acute Leukaemia French Association (ALFA) 9000 protocol, survivin expression was significantly associated with a lower WBC (P = 0·03 by the Mann–Whitney test) and favourable/intermediate cytogenetics (P = 0·03 by the Fisher test). There was no significant difference in complete remission rate or overall survival between survivin‐positive and survivin‐negative AML patients (P = 0·15 by the log‐rank test). However, survivin expression became an independent negative prognostic factor for survival when adjusted with the Cox model for established prognostic factors in AML (cytogenetics, age and WBC) or for the ALFA 9000 treatment arm (RR = 2·8 and P = 0·026, by the likelihood‐ratio test). These data suggest that survivin expression may be considered as a new unfavourable prognostic factor of de novo AML and suggest a role for apoptosis inhibition in influencing disease outcome.

Myeloid Sarcoma: Clinical and Morphologic Criteria Useful for Diagnosis

Extramedullary accumulation of myeloblasts or immature myeloid cells form tumors called myeloid sarcoma in the WHO classification. Such tumors develop in lymphoid organs, bone (skull, orbit, etc.), skin, soft tissue, various mucosae and organs, and the CNS. They may precede or occur concurrently with acute myeloid leukemia, or reveal blastic transformation of chronic myeloproliferative disorders or myelodysplastic syndromes. They may also reveal relapses in treated patients. They are constituted by a diffuse infiltrate made up of medium-to-large cells. The cells are difficult to identify. Imprints are very useful. Immunohistochemistry can help diagnose and distinguish four variants: granulocytic myeloperoxidase (MPO+, CD 68+ [KP1, PGM1-] lysozyme+, CD 34), monoblastic (MPO-, CD 68+, [KPI+, PGMI+] lysozyme+, CD 34-), myelomonoblastic (MPO-, CD 68+, [KPI+, PGM1+] lysozyme+, CD 34-), or megakaryoblastic (positivity for factor VIII, CD 61, CD 31). Immunohistochemistry sometimes demonstrates expression of CD 43, CD 7, CD 79a, and CD 56 (particularly the monoblastic variant with t[8;2 1]). Recently the demonstration of CD 99 and CD 1 7, which can now be done on paraffin sections, may be useful to identify blasts of granulocytic origin. The diagnosis is missed in about 50% of cases when immunohistochemistry is not used. Patients with myeloid sarcomas should be treated in the same way as patients with acute myeloblastic leukemia. Disease progression and prognosis are similar for the two conditions.

Patterns of bone marrow involvement in 58 patients presenting primary splenic marginal zone lymphoma with or without circulating villous lymphocytes

Summary. We studied 86 bone marrow biopsies (BMB) from 58 patients presenting with primary splenic marginal zone lymphoma (PSMZL). In 42 patients, a splenectomy was performed which enabled a histopathological diagnosis. In these patients, 44 biopsies were carried out before, and 25 after, splenectomy. In 16 recently observed patients, 17 BMB led to PSMZL diagnosis, and these patients were treated without splenectomy. Seven different patterns of infiltrates were recognized: intravascular, interstitial, nodular, massive, plasmacytic mimicking myeloma and transformation into large B‐cell lymphoma (DLBCL). The association of an intravascular infiltrate and nodules with a germinal centre and/or a marginal zone favoured a diagnosis of MZL. Immunohistochemistry demonstrated the expression of B cell‐associated antigens and, in 40% of the patients, a monotypic lymphoplasmacytic cell component. These patients often presented a serum M component and autoimmune disorders. In the past, such cases have been diagnosed as lymphoplasmacytic lymphoma. BM involvement was present in all patients. Successive biopsies showed progression and, after chemotherapy, a slight decrease in infiltrates. Transformation into DLBCL occurred in 11 of 34 patients. The patterns described are not specific for PSMZL and occur also in primary nodal MZL and, more rarely, in MALT‐type lymphoma.