Interferon-alpha induces apoptosis in human KB cells through a stress-dependent mitogen activated protein kinase pathway that is antagonized by epidermal growth factor.

We have demonstrated that interferon-alpha2-recombinant (IFNalpha) at growth inhibitory concentrations enhances the expression and signalling activity of the epidermal growth factor receptor (EGF-R) in human epidermoid carcinoma KB cells. Here we report that KB cells exposed to IFNalpha underwent apoptotic cell death and this effect was antagonized by EGF. We have also found that IFNalpha enhanced the expression of heat shock proteins (HSP) HSP-70, HSP-90 and HSP-27 and activated the NH2-terminal Jun kinase-1 (JNK-1) and p38 mitogen activated protein kinase, the target enzymes of a stress-dependent intracellular transduction pathway. Moreover, the overexpression of the wild-type JNK-1, obtained through plasmid transfection of KB cells, induced apoptosis which was potentiated by the exposure of wild-type JNK-1 (JNK-1wt)-transfected cells to IFNalpha. All these effects were neutralized by the addition of EGF to parental and JNK-1wt-transfected KB cells exposed to IFNalpha. In conclusion, EGF has a protective effect on KB cells from apoptosis while antagonizing a stress response elicited by IFNalpha and targeted on the stress pathway terminal kinases.

Desferioxamine increases iron depletion and apoptosis induced by ara-C of human myeloid leukaemic cells.

We investigated whether changes in iron metabolism and the transferrin receptor (TRF-R) expression were involved in the antileukaemic effects of arabinoside cytosine (ara-C). Treatment with 100 nM ara-C for 48h reduced thymidine uptake and increased the surface expression of the TRF-R on leukaemic blasts derived from 13/16 (81%) patients and on the HL-60 and U-937 cell lines. Whereas intracellular non-haem iron was strongly depleted 24 h after ara-C addition, TRF-R up-regulation and recovery of intracellular non-haem iron concentration occurred together after a longer exposure of the cultured cells to the drug. Since iron is an essential regulator of cell proliferation we have evaluated the effects of the combination between ara-C and the iron chelator desferioxamine (DSF) on the growth of HL-60 and U-937 cells. We found that desferioxamine strongly potentiated the effects of ara-C on leukaemic cell growth inhibition and apoptosis. This is the first report of a positive interaction between ara-C and an iron chelator in terms of antileukaemic effects.

Transient exposure to cytarabine increases peptide growth factor receptor expression and tumorigenicity of melanoma cells.

We have demonstrated that anticancer drugs at cytostatic concentrations enhance the expression and function of epidermal growth factor (EGF-R) and transferrin (TRF-R) receptors on human tumor cells. We hypothesized that these effects could represent a protective response of tumor cells to sublethal antiproliferative stimuli which could lead to enhanced growth potential. 72 hours exposure of human melanoma GLL-19 cells to 1,000 nM ara-C induced growth inhibition and increased the number of EGF-R, TRF-R and nerve growth factor receptor (NGF-R) on cell surface. Enhanced expression of beta 3 integrins CD49a, CD49c and CD49e, av integrin CD51, beta 3 integrin CD61, CD58/LFA3 and collagen IV and laminin was also detected in ara-C-treated GLL-19 cells. These changes at the tumor cell surface were paralleled by increased in vitro adhesion, invasive potential and clonogenic growth in soft agar and in vivo tumor formation. A more aggressive tumor cell phenotype is induced in human melanoma cells after transient exposure to cytostatic concentrations of ara-C.

Interferon alpha2 recombinant and epidermal growth factor modulate proliferation and hypusine synthesis in human epidermoid cancer KB cells.

We previously found that interferon alpha2 recombinant (IFNalpha) increases the expression of epidermal growth factor receptor (EGF-R) in the human epidermoid cancer KB cell line. Here we report the effects of IFNalpha and epidermal growth factor (EGF) on KB cell cycle kinetics. IFNalpha (1000 i.u./ml) for 48 h decreased the S-phase fraction and diminished the expression of Ki67 and proliferating cell nuclear antigen on KB cells. Incubation of IFNalpha-treated KB cells with 10 nM EGF for 12 h reversed these effects. We then studied several biochemical markers of cell proliferation. Ornithine decarboxylase activity was decreased to about one-tenth by IFNalpha and partly restored by EGF. Hypusine is contained only in eukaryotic initiation factor 5A and its levels are correlated with cell proliferation. IFNalpha decreased hypusine synthesis by 75%; exposure of cells to EGF for 12 h restored hypusine synthesis almost completely. We also studied the effects of IFNalpha on the cytotoxicity of the recombinant toxin TP40, which inhibits elongation factor 2 through EGF-R binding and internalization. IFNalpha greatly enhanced the TP40-induced inhibition of protein synthesis in KB cells. In conclusion, IFNalpha, which affects protein synthesis machinery and increases EGF-R expression, enhances the tumoricidal activity of TP40 and hence could be useful in the setting of anti-cancer therapy.

alpha-Interferon potentiates the growth inhibitory effects of anti-transferrin receptor monoclonal antibodies.

We have demonstrated that interferon-alpha 2 recombinant (IFN alpha) inhibits the growth and modulates the expression of the receptor for transferrin (TRF-R) in human epidermoid carcinoma KB cells. Receptor upregulation results in the reconstitution of intracellular iron levels in the IFN alpha-treated cells. Several anti-TRF-R murine monoclonal antibodies (MAbs) have been generated which induce tumor cell growth inhibition through blockade of receptor function. We have evaluated by MTT assay the effect of anti-TRF-R 42/6, E2.3, A27.15 and D65.30 MAbs given in combination with IFN alpha on the growth of human epidermoid carcinoma KB cells. We found that IFN alpha and A27.15 MAb induced a synergistic antiproliferative effect on these cells. These results suggest that IFN alpha may potentiate the antitumor efficacy of TRF-R-targeted therapy.

alpha-Interferon potentiates epidermal growth factor receptor-mediated effects on human epidermoid carcinoma KB cells.

The molecular mechanisms underlying the growth inhibition of human tumor cells induced by recombinant interferon-alpha (IFN alpha) are mostly unknown. It has been proposed that this effect could be related to down-regulation and/or impaired function of peptide growth factor receptors (PGF-Rs) in tumor cells exposed to IFN alpha. However, we have previously described that IFN alpha-induced growth inhibition of human epidermoid carcinoma cells is paralleled by up-regulation of epidermal growth factor receptor (EGF-R). Here we report that an increase in EGF-R synthesis is detectable after 3 hr of exposure to cytostatic concentration of IFN alpha in epidermoid KB tumor cells. In these experimental conditions IFN alpha does not depress and even potentiates EGF-R function. IFN alpha-treated KB cells retain sensitivity to the cytotoxic activity of the anti-EGF-R 225 monoclonal antibody (MAb), which acts through receptor blockade, and are sensitized to the growth-promoting effect of EGF. EGF-induced tyrosine (tyr) phosphorylation both of total cellular protein extracts and of the immunoprecipitated EGF-R is increased in IFN alpha-treated cells. We conclude that a cross-talk between IFN alpha and EGF occurs in KB cells since IFN alpha, at cytostatic concentration, potentiates the effects mediated by the EGF-R.

5-Aza-2'-deoxycytidine induces growth inhibition and upregulation of epidermal growth factor receptor on human epithelial cancer cells.

BACKGROUND: The epidermal growth factor (EGF-R) receptor is an important growth regulator of epithelial cancer cells, and is presently considered a tumor-associated antigen (TAA) which is overexpressed by several human cancers and barely detectable in most normal tissues. Since TAA density at the tumor cell surface is a critical factor regulating the efficiency of immunotargeting procedures, a therapeutic advantage may derive from the pharmacologic enhancement of membrane expression of such antigens on tumor cells. MATERIALS AND METHODS: Utilizing a panel of different human cancer cell lines of epithelial derivation, we have investigated in the in vitro effects of 5-aza-2'-deoxycytidine (5azaCdR), an antineoplastic agent able to induce gene activation and phenotypic modulation, on the surface expression of EGF-R by tumor cells. RESULTS: 5azaCdR (10-1000 nM) induced growth inhibition, in the absence of acute cell kill, on KB (human oropharyngeal carcinoma), LoVo and the drug-resistant clone LoVo-DX (colon carcinoma) and A549 (lung adenocarcinoma) cell lines, along with a significant enhancement of EGF-R expression at the tumor cell surface. A single 24 h pulse of 5azaCdR, followed by 96 h of culture in drug-free medium, induced 50% growth inhibition on KB cells at a concentration (IC50) of 500 nM, on A549 (IC50 = 490 nM), LoVo (IC50 = 400 nM) and LoVo-DX (IC50 = 100 nM) cell lines. Under these conditions the specific binding of 125I-EGF was significantly upregulated at the surface of growth-inhibited cancer cells. Scatchard analysis of EGF-binding data revealed no changes in the Kd of EGF-R for its ligand in 5azaCdR-treated tumor cells and demonstrated a significant increase in the number of both the high- and low-affinity EGF-binding sites on KB cells, while only one class of EGF-binding site was detectable on A549, LoVo and LoVo-DX tumor cell lines before and after exposure to 5azaCdR. The EGF-R upregulation induced by 5azaCdR was paralleled by the increased binding of the anti-EGF-R monoclonal antibody (MAb) 108.1 on the surface of cancer cells. Finally, the rate of endocytosis of the anti-EGF-R MAb by KB cells was not modified by drug treatment, indicating that exposure to 5azaCdR does not hamper MAb internalization by the tumor cells. This latter represents an essential process for the cytotoxic effects of immunoconjugate drugs or toxins. CONCLUSIONS: We suggest a role for 5azaCdR in enhancing the efficacy of therapeutic approaches involving the use of anti-EGF-R immunoconjugated for the imaging and the treatment of human epithelial neoplasias.