The nuclear aryl hydocarbon receptor is involved in regulation of DNA repair and cell survival following treatment with ionizing radiation.

In the present study, we explored the role of the aryl hydrocarbon receptor (AhR) for γ-H2AX associated DNA repair in response to treatment with ionizing radiation. Ionizing radiation was able to stabilize AhR protein and to induce a nuclear translocation in a similar way as described for exposure to aromatic hydrocarbons. A comparable AhR protein stabilization was obtained by treatment with hydroxyl-nonenal-generated by radiation-induced lipid peroxidation. AhR knockdown resulted in significant radio-sensitization of both A549- and HaCaT cells. Under these conditions an increased amount of residual γ-H2AX foci and a delayed decline of γ-H2AX foci was observed. Knockdown of the co-activator ARNT, which is essential for transcriptional activation of AhR target genes, reduced AhR-dependent CYP1A expression in response to irradiation, but was without effect on the amount of residual γ-H2AX foci. Nuclear AhR was found in complex with γ-H2AX, DNA-PK, ATM and Lamin A. AhR and γ-H2AX form together nuclear foci, which disappear during DNA repair. Presence of nuclear AhR protein is associated with ATM activation and chromatin relaxation indicated by acetylation of histone H3. Taken together, we could show, that beyond the function as a transcription factor the nuclear AhR is involved in the regulation of DNA repair. Reduction of nuclear AhR inhibits DNA-double stand repair and radiosensitizes cells. First hints for its molecular mechanism suggest a role during ATM activation and chromatin relaxation, both essential for DNA repair.

Radioprotection of normal tissue to improve radiotherapy: the effect of the Bowman Birk protease inhibitor.

Specific radioprotection of normal tissue represents a promising approach to improve radiotherapy. The ultimate feature of a normal tissue selective radioprotector is that tumor tissue is excluded from protection. Radioprotectors of the current generation, such as Ethyol, are not explicit normal tissue specific. In contrast, the Bowman Birk protease inhibitor, which is known to prevent in vitro and in vivo radiation-induced carcinogenesis, was found to be normal tissue specific. Moreover, the molecular restrictions for this specificity were identified. The radioprotective effect is dependent upon the presence of a functional wt. TP53. Since a high amount of tumors have lost TP53 function during tumor development, the clinical application of BBI to protect normal tissue from radiation damage would effectively improve the therapeutic outcome of radiation therapy. We succeeded to identify stimulation of DNA-repair mechanisms, such as nucleotide excision repair (NER) and nonhomologous end joining (NHEJ), as molecular mode of action. These results are in good agreement with the observations that BBI concomitantly exhibits anticarcinogenic effect and radioprotective effects. Taken together, BBI is recommended as a radioprotector for normal tissue expressing wild type TP53 during treatment of tumors characterized by a mutant TP53.

Characterization of the amino acids essential for the photo- and radioprotective effects of a Bowman-Birk protease inhibitor-derived nonapeptide.

The Bowman-Birk protease inhibitor has been reported to exert photo- and radioprotective activity. This effect was assigned to a cyclic nonapeptide sequence which is known to contain the amino acids responsible for the anti-chymotryptic activity of the BBI. The present study indicated that linearization of the nonapeptide resulted in a significant loss of anti-proteolytic activity, whereas the photo- and radioprotective capacity persisted. Substitution of the amino acids Leu or Ser of the nonapeptide, essential for the anti-proteolytic activity, with different amino acids, indicated that rather the hydrophobic features of the amino acids in this position than charge are critical to retain the photo- and radioprotective effect. These results suggest the existence of a bifunctional peptide sequence with anti-proteolytic and photo-/radioprotective capacity. However, the lack of correlation between the photo-/radioprotective activity and the anti-proteolytic activity within the peptides generated by modification of the linear nonapeptide argues for the existence of two closely colocalized domains within the nonapeptide responsible for photo-/radioprotection and protease inhibition.

O-phospho-L-tyrosine protects TP53 wild-type cells against ionizing radiation.

O-phospho-L-tyrosine (P-Tyr) has been reported previously to inhibit growth of several cancer cell lines at mM concentrations. In the present study, we investigated the effect of this compound on tumor cells and normal cells in combination with radiation exposure. It could be demonstrated for the first time that P-Tyr at microM concentrations protects TP53 wild-type cells against ionizing radiation (SF4 minus BBI = 0.28, SF4 plus BBI = 0.45). On the contrary, human transformed or tumor cell lines characterized by mutated or functional inactivated TP53 were not altered or increased in their radiation sensitivity (SF4 minus BBI = 0.32, SF4 plus BBI = 0.22). Treatment of wild-type TP53 cells with P-Tyr induced stabilization of TP53 within 3 and 16 hours and a subsequent increase in CDKN1A expression after treatment. Consequently, a 16-hours pretreatment of cells with P-Tyr led to a significant radioprotective effect. This was not observed in cell lines with mutated TP53, which shows no radioprotection by P-Tyr. Thus, the present data suggest that P-Tyr-mediated radioprotection is dependent on preirradiation stabilization of TP53. The results indicate that P-Tyr is a radioprotective agent that can potentially be very useful and easy to deliver for radiation protection in general and especially in radiation therapy of TP53-mutated tumors.

The Bowman-Birk protease inhibitor enhances clonogenic cell survival of ionizing radiation-treated nucleotide excision repair-competent cells but not of xeroderma pigmentosum cells.

PURPOSE: The radioprotective effect of the Bowman-Birk protease inhibitor (BBI) was previously shown to result from a TP53 dependent mechanism. Whether this effect involves specific DNA repair mechanisms is now tested. MATERIAL AND METHODS: Normal human fibroblasts were pre-treated with BBI before exposure to X-rays, UVB or to chemical agents (bleomycin, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), cisplatin). These agents were chosen because of their ability to induce different spectra of DNA damage. The radiometric agent bleomycin primarily induces double-strand breaks (dsb), which are repaired by recombination; MNNG results in alkylated bases which are repaired by base excision repair (BER); cisplatin results in DNA-crosslinks which are repaired mainly by nucleotide excision repair (NER); and finally UVB generates thymine dimers and thymine-cytosine-6-4 products which are also repaired by NER. Cell survival was analysed by colony formation assay and DNA dsb by constant field gel electrophoresis. The combined effect of BBI and X-rays was also tested for XP-fibroblasts, which are defective in NER. RESULTS: For normal human fibroblasts the radioprotective effect of BBI was clearly found by using a delayed plating procedure. The radioprotective effect was found to be unrelated to an altered induction or repair of radiation-induced DNA dsb. Pretreatment with BBI did not affect cell killing after exposure to bleomycin or MNNG, but resulted in a significant protection of cells exposed to cisplatin or UVB. These results indicate that pre-treatment with BBI did not alter recombination repair or BER, but was able to modify NER. The latter finding was supported by the observation made for XP-cells, where pretreatment with BBI failed to result in radioprotection after exposure to ionizing radiation. CONCLUSIONS: On the basis of these data it is proposed that the radioprotective effect of BBI is the result of an improved nucleotide excision repair mechanism.

The presence of wild-type TP53 is necessary for the radioprotective effect of the Bowman-Birk proteinase inhibitor in normal fibroblasts.

In the present study we have demonstrated that the Bowman-Birk proteinase inhibitor (BBI) protected normal fibroblasts from a radiation-induced reduction in cell survival, whereas in transformed fibroblasts no radioprotective effect was observed. It was shown that BBI reduced the radiation-induced protein stabilization and DNA-binding activity of TP53 (formerly known as p53) in normal fibroblasts. In transformed fibroblasts, BBI failed to induce these effects. The analysis of the TP53 gene in transformed fibroblasts revealed a mutation in exon 5. As a consequence of this mutation, the expression of the TP53 downstream gene CDKN1A (p21/WAF1/Cip1) is blocked. Based on experiments using TP53 antisense oligonucleotides, the radioprotective effect of BBI could be correlated with the function of wild-type TP53. Thus BBI can be considered as a selective radioprotective agent for normal human fibroblasts.

The radioprotective effect of BBI is associated with the activation of DNA repair-relevant genes.

PURPOSE: To investigate the molecular mechanisms of the radioprotective effect of the Bowman-Birk proteinase inhibitor (BBI) in normal human skin fibroblasts (HSF). MATERIAL AND METHODS: The effect of BBI pre-treatment on p53 protein level and on mRNA levels of downstream genes (ERCC3, Gadd45 and p53) was investigated. RESULTS: As indicated by time-course experiments based on clonogenic assays, a 6 h pre-incubation with BBI before irradiation of HSF with a single dose of 6 Gy resulted in maximum radioprotection. In non-irradiated cells, pre-incubation with BBI resulted in an increased level of p53 protein. Concomitantly, enhanced mRNA levels of the ERCC3 and the Gadd45 genes were observed. As a consequence, BBI-treated cells showed accelerated DNA repair compared with untreated cells when irradiated. CONCLUSIONS: The radioprotective effect of the Bowman-Birk proteinase inhibitor was accompanied by elevated mRNA expression of repair-relevant genes prior to irradiation. Activation of the DNA-repair machinery induced by pre-treatment with BBI is one possible mechanism of the radioprotective effect of BBI.

MK-886, a leukotriene biosynthesis inhibitor, induces antiproliferative effects and apoptosis in HL-60 cells.

MK-886, a specific inhibitor of 5-lipoxygenase inhibited DNA replication in leukemic HL-60 cells in a dose-dependent manner. Addition of exogenous leukotriene B4 reversed this effect, whereas addition of leukotriene B4 failed to modulate a prostaglandin D2-induced inhibition of DNA replication. The reversal of MK-886-induced inhibition was not observed with leukotriene C4. These results suggest that the effect of MK-886 is mediated by inhibition of leukotriene B4 biosynthesis. Moreover, MK-886 not only impaired DNA replication in HL-60 cells but also decreased cell proliferation and induced apoptotic cell death. Our results suggest a crucial role of leukotriene B4 in the regulation of cell proliferation and cell survival in HL-60 cells.

HL-60 leukemia cells produce an autocatalytically truncated form of matrix metalloproteinase-9 with impaired sensitivity to inhibition by tissue inhibitors of metalloproteinases.

92-kDa type IV collagenase/gelatinase (matrix metalloproteinase-9; MMP-9; gelatinase B) expression and secretion has been shown to correlate with the invasive and metastatic potential of various malignant cells. MMP activity is tightly controlled by specific tissue inhibitors of metalloproteinases (TIMPs). We found the leukemic cell line HL-60 constitutively to release a 94-kDa gelatinase which we identified as MMP-9 shortened by nine amino acids at its N-terminal end. An additional gelatinolytic activity was present in small amounts and identified as a 63-kDa fragment of MMP-9 generated by autocatalytical processing. Both enzymes were identical regarding their N-terminus, indicating C-terminal truncation for the former. Incubation of cells with phorbol ester resulted in elevated amounts of both enzymes in conditioned media and in the secretion of TIMP-1. Both gelatinases were shown to be activated by trypsin and organomercurials and to possess similar activities towards various substrates. However, the 63-kDa enzyme differed from the 94-kDa enzyme in a significantly reduced inhibition by recombinant TIMP-1 and TIMP-2. Thus, the 63-kDa fragment of MMP-9 once activated may escape the regulatory influence of its specific inhibitors and may thereby promote matrix degradation during invasion of leukemic cells.

Leukemic cells (HL-60) produce a novel extracellular matrix-degrading proteinase that is not inhibited by tissue inhibitors of matrix metalloproteinases (TIMPs).

In addition to the known 94-kd gelatinase (matrix metalloproteinase 9, MMP-9), HL-60 leukemia cells release a hither-to undescribed 45-kd metalloproteinase into the culture medium. This enzyme cleaves the synthetic substrate Pro-Gln-Gly-Ile-Ala-Gly-Gln-Arg, which represents the cleavage site for collagenases in collagen type I not between isoleucine and alanine--the typical cleavage site for collagenases--but between alanine and glycine. The enzymatic activity was purified through a combination of zinc-chelate-Sepharose column chromatography, precipitation with Fractogel TSK-AF Red and gelatin-Sepharose, and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Microsequence analysis of the NH2-terminus of the purified 45-kd proteinase revealed the sequence Asp-Ile-Ser-Lys-Tyr-Thr-Thr-Thr-, which could not be found in other proteins when searched in several protein data bases. Incubation of the enzyme immobilized on nitrocellulose membranes with polyclonal antibodies to collagenase and stromelysin or gelatinases revealed no cross-reactivity. The proteolytic activity was not increased by treatment with trypsin, 8M urea, acid, or organomercurials. The proteinase, which was inhibited by chemical inhibitors of metalloproteinases, such as phenanthrolene or EDTA, is able to degrade several matrix constituents, such as collagen type IV, fibronectin, gelatin, and proteoglycans. In contrast to all known MMPs, the proteolytic activity of the 45-kd enzyme was not abolished upon incubation with recombinant tissue inhibitors of matrix metalloproteinases (TIMP) 1 or 2. Thus, the novel enzyme may influence extracellular matrix (ECM) turnover in vivo because its activity is not influenced by specific inhibitors of MMPs.

A 41 kDa transferrin related molecule acts as an autocrine growth factor for HL-60 cells.

HL-60 cells produce an autostimulatory growth factor. Since the stimulatory effect of HL-60 conditioned medium is only observed in the absence of exogenous transferrin we have assayed HL-60 cells for the production of transferrin and found that they produce polypeptides which react with transferrin antibodies. 35S-methionine labelling, immunoprecipitation and subsequent separation by SDS-gel electrophoresis reveals the presence of a major transferrin related 41 +/- 2 kDa species released by HL-60 cells. Physiological levels of iron salts completely abolish the requirement of exogenous transferrin which indicates that the endogenous transferrin related polypeptides in the presence of exogenous inorganic iron salts are sufficient for the proliferation of HL-60 cells provided insulin or related growth factors are present. The addition of transferrin receptor antibodies inhibits the stimulatory action of the endogenous transferrin related activity.

How do normal and leukemic white blood cells egress from the bone marrow? Morphological facts and biochemical riddles.

Under normal circumstances only mature granulocytes and monocytes cross the bone marrow sinus wall, a trilaminar structure consisting of endothelial cells, a discontinuous basal membrane and an adventitial cell layer in order to get access to the blood circulation. In leukemia, however, immature white blood cells are able to traverse the barrier and to appear in the blood stream. Very little is known about the regulatory processes which govern the egress of white blood cells in healthy individuals and their malignant counterparts in patients with leukemia. The results of the few studies performed to address this question in animal and human leukemias all agree that the extent to which adventitial cells (fibroblasts) cover the endothelium in bone marrow is drastically reduced. This implies altered interactions between the leukemic and adventitial cells and their extracellular matrix. We propose here a model to explain the egress of normal cells and their leukemic counterparts. It is based upon our own experimental data and the general at present limited knowledge of the subject. It is hoped that this model will stimulate further research into this important aspect of leukemogenesis.