Interaction of HSV-1 infected peripheral blood mononuclear cells with cultured dermal microvascular endothelial cells: a potential model for the pathogenesis of HSV-1 induced erythema multiforme.

The effect of herpes virus infection on human dermal microvascular endothelial cells and herpes-virus-1-infected peripheral blood mononuclear cells on human dermal microvascular endothelial cells was studied as a model of herpes-associated erythema multiforme. After infection of human dermal microvascular endothelial cells with native herpes virus and overnight culture, 60%--90% of human dermal microvascular endothelial cells showed cytopathic effects. HLA class I molecules and CD31 (PECAM-1) surface expression in herpes-virus-infected endothelial cells were substantially downregulated, whereas CD54 (ICAM-1) remained unchanged. Cocultivation with herpes-virus-1-infected peripheral blood mononuclear cells left characteristic plaques on the human dermal microvascular endothelial cell monolayer; however, very few human dermal microvascular endothelial cells (1%--3%) were infected. Adhesion molecule expression of human dermal microvascular endothelial cells cocultivated with herpes-virus-infected peripheral blood mononuclear cells demonstrated a 5-fold increase in CD54 expression, a 2-fold increase in HLA class I expression, but no change of CD31 by fluorescence-activated cell sorter analysis. Incubation of human dermal microvascular endothelial cells with ultraviolet-C irradiated herpes-virus-infected peripheral blood mononuclear cells had no effect on morphology or adhesion molecule expression levels. Changes of adhesion molecule expression by direct infection or cocultivation with peripheral blood mononuclear cells (with native and ultraviolet-C inactivated herpes virus infection) were also documented at the mRNA level. Adhesion assays demonstrated an increased binding of herpes-virus-infected peripheral blood mononuclear cells versus noninfected peripheral blood mononuclear cells to noninfected human dermal microvascular endothelial cells. Our results suggest that incubation of herpes-virus-infected peripheral blood mononuclear cells with human dermal microvascular endothelial cells induces significant upregulation of CD54 and major histocompatibility complex class I molecules in the surrounding noninfected human dermal microvascular endothelial cells, which is associated with an increased binding of peripheral blood mononuclear cells. Our in vitro findings may serve as a model for herpes-associated erythema multiforme possibly explaining the dermal inflammatory reaction seen in that condition.

Bcl-2 interferes with the execution phase, but not upstream events, in glucocorticoid-induced leukemia apoptosis.

Due to their growth arrest- and apoptosis-inducing ability, glucocorticoids (GC) are widely used in the therapy of various lymphoid malignancies. Cell death is associated with activation of members of the interleukin-1beta-converting enzyme (ICE) protease/caspase family and, is presumably prevented by the anti-apoptotic protein Bcl-2. To further address the role of Bcl-2 in GC-mediated cytotoxicity, we generated subclones of the GC-sensitive human T-cell acute lymphoblastic leukemia line CCRF-CEM, in which transgenic Bcl-2 expression is regulated by tetracycline. Up to about 48 h, exogenous Bcl-2 almost completely protected these cells from apoptosis, digestion of poly-ADP ribose polymerase (PARP) and generation of Asp-Glu-Val-Asp cleaving (DEVDase) activity. However, when the cells were cultured for another 24 h in the continuous presence of GC, they underwent massive apoptosis that was associated with DEVDase activity and PARP cleavage. Bcl-2 did not markedly affect GC-mediated growth arrest, thereby separating the anti-proliferative from the apoptosis-inducing effect of GC. Moreover, Bcl-2 did not prevent the dramatic reduction in the levels of several mRNAs observed during GC treatment, including the transgenic Bcl-2 mRNA. Thus, Bcl-2 can be placed upstream of effector caspase activation, but downstream of other GC-regulated events, such as growth arrest and the potentially critical repression of steady state levels of multiple mRNA.

Irradiation induces G2/M cell cycle arrest and apoptosis in p53-deficient lymphoblastic leukemia cells without affecting Bcl-2 and Bax expression.

The tumor suppressor p53 has been implicated in gamma irradiation-induced apoptosis. To investigate possible consequences of wild-type p53 loss in leukemia, we studied the effect of a single dose of gamma irradiation upon p53-deficient human T-ALL (acute lymphoblastic leukemia) CCRF - CEM cells. Exposure to 3 - 96 Gy caused p53-independent cell death in a dose and time-dependent fashion. By electron microscopic and other criteria, this cell death was classified as apoptosis. At low to intermediate levels of irradiation, apoptosis was preceded by accumulation of cells in the G2/M phase of the cell division cycle. Expression of Bcl-2 and Bax were not detectably altered after irradiation. Expression of the temperature sensitive mouse p53 V135 mutant induced apoptosis on its own but only slightly increased the sensitivity of CCRF - CEM cells to gamma irradiation. Thus, in these, and perhaps other leukemia cells, a p53- and Bcl-2/Bax-independent mechanism is operative that efficiently senses irradiation effects and translates this signal into arrest in the G2/M phase of the cell cycle and subsequent apoptosis.

p53-induced apoptosis in the human T-ALL cell line CCRF-CEM.

The tumor suppressor p53 has been implicated in apoptosis induction and is mutated in human T-ALL CCRF-CEM cells. To investigate possible consequences of wild-type p53 loss, we reconstituted CEM-C7H2, a subclone of CCRF-CEM, with a temperature-sensitive p53 allele (p53ts). Stably transfected lines expressed high levels of p53ts and shift to the permissive temperature (32 degrees C) caused rapid induction of p53-regulated genes, such as p21(CIP1/WAF1), mdm-2 and bax. This was followed by extensive apoptosis within 24 h to 36 h, supporting the notion that mutational p53 inactivation contributed to the malignant phenotype. p53-dependent apoptosis was preceded by digestion of poly(ADP-ribose) polymerase, a typical target of interleukin-1beta-converting enzyme (ICE)-like proteases/caspases, and was markedly resistant to the ICE/caspase-1 and FLICE/caspase-8 inhibitor acetyl-Tyr-Val-Ala-Asp.chloromethylketone (YVAD), but sensitive to the CPP32/caspase-3 inhibitor benzyloxycarbonyl-Asp-Glu-Val-Asp.fluoromethylketone (DEVD) and benzyloxycarbonyl-Val-Ala-Asp.fluoromethylketone (zVAD), a caspase inhibitor with broader specificity. This indicated an essential involvement of caspases, but argued against a significant role of ICE/caspase-1 or FLICE/caspase-8. Actinomycin D or cycloheximide prevented cell death, suggesting that, in this system, p53-induced apoptosis depends upon macromolecule biosynthesis. Introduction of functional p53 into CEM cells enhanced their sensitivity to the DNA-damaging agent doxorubicin, but not to the tubulin-active compound vincristine. Thus, mutational p53 inactivation in ALL might entail relative resistance to DNA-damaging, but not to tubulin-destabilizing, chemotherapy.

Splice site mutation in the glucocorticoid receptor gene causes resistance to glucocorticoid-induced apoptosis in a human acute leukemic cell line.

Induction of apoptosis is the molecular basis for the therapeutic application of glucocorticoids (GC) in human leukemia. The beneficial effect of endocrine therapy is, however, hampered by the occurrence of resistant clones evolving under selective GC pressure. To delineate molecular mechanisms of GC resistance, we PCR amplified, cloned, and sequenced GC receptor (GR) transcripts and gene segments from a GC-resistant subclone of the human acute leukemic cell line CCRF-CEM, termed CEM-R6. Our analyses revealed that one GR gene allele harbored a point mutation (L753F) previously shown to compromise GR functions in other CCRF-CEM derivatives. On the second allele, we identified an A to G point mutation in the 3'-splice junction of intron G. As a consequence, a cryptic splice site 8 base pairs downstream within exon 8 is recognized, which leads to an 8-base deletion in the GR mRNA, resulting in reading frame shift and 2 consecutive in-frame preterminal stop codons. Translation of this mutant mRNA would produce a truncated GR protein missing 93 amino acids of the ligand-binding domain and expressing 9 altered residues at its new COOH terminus. In concert with the L753F mutation on the other allele, this molecular defect explains the GC-resistant phenotype and provides further evidence for mutational GR gene inactivation as a mechanism for human leukemic cells to escape GC-induced apoptosis.