A highly conserved proapoptotic gene, IKIP, located next to the APAF1 gene locus, is regulated by p53.

We describe here the identification and initial characterization of a novel human gene termed IKIP (I kappa B kinase interacting protein) that is located on chromosome 12 in close proximity to APAF1 (apoptotic protease-activating factor-1). IKIP and APAF1 share a common 488 bp promoter from which the two genes are transcribed in opposite directions. Three IKIP transcripts are generated by differential splicing and alternative exon usage that do not show significant homology to other genes in the databases. Similar to APAF1, expression of IKIP is enhanced by X-irradiation, and both genes are dependent on p53. Moreover, IKIP promotes apoptosis when transfected into endothelial cells. We conclude that IKIP is a novel p53 target gene with proapoptotic function.

Oligodeoxynucleotides containing CpG motifs induce low levels of TNF-alpha in human B lymphocytes: possible adjuvants for Th1 responses.

Oligodeoxynucleotides containing CpG motifs (CpG-ODN) represent potential adjuvants for specific immunotherapy of type I allergies because they foster Th1-like immune responses. However, previous work has shown that CpG-ODN induce systemically active levels of TNF-alpha in murine macrophages. The goal of the present study was to evaluate the release of TNF-alpha in human cells by a CpG-ODN proven to induce Th1 immune responses in cells from atopic individuals and in mice. CpG-ODN induced TNF-alpha in cells from atopic and healthy individuals. However, the amounts were low, as determined by comparison with commonly used Ags. Intracellular cytokine staining of PBMC revealed that CpG-ODN-induced TNF-alpha derived exclusively from B lymphocytes. TNF-alpha contributed to the CpG-ODN-augmented proliferation and Ig synthesis in PBMC, but was not involved in IFN-gamma synthesis. In conclusion, our findings indicate that certain CpG-ODN induce low amounts of TNF-alpha in human B lymphocytes and may therefore be used to modulate Th2-biased immune responses in allergic patients.

Characterisation of genotoxic properties of 2',2'-difluorodeoxycytidine.

The genotoxic properties of 2',2'-difluorodeoxycytidine (dFdC) were characterised using diploid, mortal low-passage fibroblasts (LPF cells) and the spontaneously transformed fibroblast cell line V79. In both cell types, incorporation of dFdC into the DNA led to an increase of DNA single-strand breaks evaluated by an in situ nick translation assay and to an accumulation of cells in the S-phase of the cell cycle. At concentrations below those leading to cell cycle arrest, dFdC neither induced sister chromatid exchange (SCE) nor structural chromosome aberrations in LPF cells, whereas V79 cells accumulated SCEs as well as chromosome breaks over a broad dose range. In LPF cells treated with dFdC, chromosomal alterations were detected by the micronucleus assay within a narrow concentration range, whereas in V79 cells, a dose-dependent increase in the appearance of micronuclei was seen up to cytotoxic concentrations. In addition, V79 cells went into apoptosis, as evaluated by nuclear fragmentation and condensation, whereas this phenomenon was not detectable in LPF cells.

alpha-Melanocyte stimulating hormone downregulates differentiation-driven heat shock protein 70 expression in keratinocytes.

Heat shock proteins are versatile tools engaged in several cellular functions. In particular, the stress-inducible 70-kDa heat shock protein (hsp70) not only confers protection on cells but also is involved in the regulation of the production of cellular stress response mediators including cytokines. In addition to cytokines, neurohormones such as alpha-melanocyte stimulating hormone (alphaMSH) were recently found to be potent mediators of inflammatory and immune responses. Thus, the current study was performed to investigate the role of alphaMSH in the expression of hsp70 in a human keratinocyte cell line (HaCaT). Proliferation and differentiation of HaCaT cells are known to be regulated by changing extracellular Ca2+ concentrations. HaCaT cells induced to differentiate in high Ca2+ medium (1.5 mM) were found to express higher levels of hsp70 protein than cells grown under low Ca2+ conditions. Moreover, differentiated HaCaT cells were markedly more resistant to oxidative stress than undifferentiated control cells. alphaMSH significantly suppressed hsp70 expression in a concentration-dependent manner in differentiated HaCaT cells but had only a minor effect on undifferentiated cells. Upon treatment with alphaMSH, HaCaT cells grown in high Ca2+ medium were rendered more sensitive to oxidative stress, which significantly decreased their survival rate. These findings indicate that alphaMSH, which is released by keratinocytes in an autocrine fashion following injurious stimuli such as tumor promoters or ultraviolet light, is able to regulate the cells' cytoprotective protein equipment.

Drug resistance against gemcitabine and topotecan mediated by constitutive hsp70 overexpression in vitro: implication of quercetin as sensitiser in chemotherapy.

Heat shock proteins have been reported to confer resistance to certain antineoplastic drugs. We investigated the impact of hsp70 overexpression on the efficacy of two new anti-cancer drugs, topotecan and gemcitabine. We used the fibrosarcoma WEHI-S cells stably transfected to overexpress the hsp70 cDNA from the constitutive SV40 promoter and appropriate control cells. After topotecan and gemcitabine treatment hsp70-overexpressing cells showed a marked elevation in cell survival, suggesting that hsp70 overexpression was sufficient to confer resistance to the drugs. In addition, hsp70-overexpressing cells were capable of starting cell proliferation after treatment with drug dosages that were lethal to control cells. Our results demonstrate that hsp70 overexpression represents a possible cause of drug resistance. In order to transfer these data to tumour cells constitutively expressing stress hsp70 due to the constitutive activity of the original hsp70 promoter we sought to supress the heat shock response pathway by the natural flavonoid quercetin, known to inactivate the heat shock transcription factor (HSF). Using a suitable cell line, we demonstrated the sensitising activity of quercetin. We found that antineoplastic drug concentrations exerting cytotoxic activity were markedly lower when cells were pretreated with quercetin. Concomitantly, hsp70 expression was strongly down-regulated under quercetin treatment. Our data indicate that quercetin may be useful as a sensitiser in chemotherapeutically treated patients suffering from hsp70-overexpressing tumours.

HSP70 overexpression mediates the escape of a doxorubicin-induced G2 cell cycle arrest.

The stress inducible heat shock protein 70 (hsp70) confers resistance to a variety of adverse environmental conditions including certain anticancer drugs. In the present study we explored cellular consequences of hsp70 overexpression with respect to genotoxic stress. Employing an isogenic set of stable transfectant cells, one overexpressing hsp70 and the other serving as control, we found that a high hsp70 expression level is sufficient to provide protection against the cytotoxicity of doxorubicin. In addition, hsp70-protected cells showed the capability to restart cell proliferation at concentrations where control cells arrested. However, the DNA lesion density was comparable in the lines used. Recording the cell cycle control revealed a dramatic shortening of the doxorubicin-mediated cell cycle arrest in the G2 phase upon hsp70 overexpression. Our data suggest an involvement of hsp70 in the regulation of the cell cycle.