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Background: In a previously published in vitro study based on top-down proteomics we found that the calcium-binding proteins S100A6 and S100A4 were affected by exposure to ionizing radiation in a p53-dependent fashion. Both proteins showed post-translational modification changes, and S100A6 also showed increased expression and translocation in response to irradiation. Aim of the present study was to evaluate the expression of S100A6 and S100A4 in non-small-cell lung cancer (NSCLC)., Methods: S100A6 expression on archival tumor cell lysates from 39 patients with radically resected NSCLC was assessed with SELDI-TOF-MS. S100A6 identity was confirmed using a SELDI-based antibody-capture method on lysates from the A549 lung cancer cell line, cell lysates from two freshly prepared NSCLC samples, four plasma samples and one pleural effusion sample. Immunostainings for S100A6, S100A4 and p53 were performed on tissue microarrays containing 103 stage I surgically resected NSCLC cases and 14 normal lung parenchyma specimens., Results: The presence of post-translationally modified S100A6 forms was confirmed with SELDI-MS on enriched tumor cell lysates, as well as in plasma and pleural effusion samples. In addition, high S100A6 peak intensity was associated with longer median survival (35 months vs. 18 months for high and low peak intensity, respectively; p=n.s.). The immunohistochemical analysis showed that 25% of tumors were S100A6 positive. S100A6 expression correlated directly with non-squamous histology (p<0.0001) and S100A4 expression (p=0.005), and inversely with p53 expression (p=0.01). S100A6-positive cases showed a trend of longer survival compared with S100A6-negative cases (p=0.07). This difference became significant when the analysis was restricted to p53-negative cases (n=72). In this subgroup of patients, whose tumors likely exhibit a functional p53, S100A6 was an independent prognostic factor of improved survival at multivariate analysis (HR 0.49, 95% CI 0.27-0.81, p=0.017)., Conclusions: In this study we have validated on clinical material our previous findings on cell lines in terms of S100A6 expression and post-translational modifications pattern in NSCLC. Moreover, the survival results obtained in p53-negative stage I NSCLC cases support the proposed pro-apoptotic function of S100A6 and suggest the hypothesis of a cross regulation between these two proteins.

Non-small cell lung carcinoma (NSCLC) is characterized by resistance to drug-induced apoptosis, which might explain the survival of lung cancer cells following treatment. Recently we have shown that the broad-range kinase inhibitor staurosporine (STS) reactivates the apoptotic machinery in U1810 NSCLC cells [Joseph et al., Oncogene 21 (2002) 65]. Lately, several STS analogs that are more specific in kinase inhibition have been suggested for tumor treatment. In this study the apoptosis-inducing ability of the STS analogs PKC 412 and Ro 31-8220 used alone or in combination with DNA-damaging agents in U1810 cells was investigated. In these cells Ro 31-8220 neither induced apoptosis when used alone, nor sensitized cells to etoposide treatment. PKC 412 as a single agent induced death of a small number of U1810 cells, whereas it efficiently triggered a dose- and time-dependent apoptosis in U1285 small cell lung carcinoma cells. In both cell types PKC 412 triggered release of mitochondrial proteins followed by caspase activation. However, concomitant activation of a caspase-independent pathway was essential to kill NSCLC cells. Importantly, PKC 412 was able to sensitize etoposide- and radiation-induced death of U1810 cells. The best sensitization was achieved when PKC 412 was administered 24 h after treatments. In U1810 cells, Ro 31-8220 decreased PMA-induced ERK phosphorylation as efficiently as PKC 412, indicating that the failure of Ro 31-8220 to induce apoptosis was not due to weaker inhibition of conventional and novel PKC isoforms. However, Ro 31-8220 increased the basal level of ERK and Akt phosphorylation in both cell lines, whereas Akt phosphorylation was suppressed in the U1810 cells, which might influence apoptosis. These results suggest that PKC 412 could be a useful tool in increasing the efficiency of therapy of NSCLC.

The aim of this article is to present a method for studying the shape of the dose and repair responses for X-ray-induced double-strand breaks (DSBs) as measured by neutral filter elution (NFE). The approach is closely related to a method we developed for the use of specific molecular size markers and used for determination of the absolute number of randomly distributed radiation-induced DSBs by pulsed-field gel electrophoresis (PFGE). Mouse leukemia L1210 cells were X-irradiated with 0-50 Gy. Samples were then evaluated both with PFGE and with NFE. Assuming that with both migration (PFGE) and elution (NFE), a heterogeneous population of double-stranded DNA fragments will start with the smallest fragments and proceed with increasingly larger fragments, it is possible to match the migration behavior of fractions of fragments smaller than a certain size to the fraction eluted at a specific time. This assumption does not exclude the possibility of DNA being sheared in the NFE filter. The yield, as determined by the size markers in PFGE, was used to find the corresponding elution times in the NFE experiment. These experimentally used elution times could then reversely be interpreted as size markers which finally were used to calculate DSBs/Mbp as a function of X-ray dose. The resulting lines were almost straight. The data were also plotted as relative elution and showed that, as expected, the dose response then appears with a more pronounced sigmoid shape.

Purpose: The DNA-PK complex is one of the major pathways by which mammalian cells respond to DNA double-strand breaks induced by ionizing radiation. This study evaluated the relationship between the immunohistochemical expression of the individual components of DNA-PK and cellular radiosensitivity in head and neck cancers., Methods and Materials: Biopsies from patients with previously untreated squamous cell carcinomas of the head and neck were assessed for inherent tumor radiosensitivity measured as the surviving fraction at 2 Gy (SF2) using a soft agar clonogenic assay. Paraffin-embedded tumor material from 64 successfully grown specimens was immunohistochemically stained for expression of DNA-PKcs and Ku (p70/p80). The same tumor material was previously analyzed for the immunohistochemical expression of p53., Results: A significant correlation was found between the degree of expression of DNA-PKcs and Ku (p70/p80) (r = 0.55, p<0.001). There were no overall significant differences in the levels of expression of DNA-PKcs and Ku (p70/p80) in tumors from patients of either sex, different sites, histologies, and stages. No relationship was found between SF2 and the expression of either DNA-PKcs (r = 0.22, p = 0.081) or Ku (p70/p80) (r = 0.064, p = 0.62). Comparison with previous immunohistochemical characterization showed no significant correlations between the expression levels of p53 and either DNA-PKcs (r = 0.093, p = 0.46) or Ku (p70/p80) (r = -0.17, p = 0.17)., Conclusions: This study suggests that determining the immunohistochemical expression of DNA-PK in head and neck cancers from multiple sites does not have a role as a predictive assay of tumor in vitro radiosensitivity.

Intrinsic radiosensitivity and rejoining of radiation-induced DNA double-strand breaks (DNA-dsb) were analysed in five lung carcinoma cell lines: U-1285, U-1906, H-69, H-82 and U-1810. RS correlated with both the initial phase of DNA-dsb rejoining, at 15 min (r2 = 0.818) and the late phase, at 120 min postirradiation (r2 = 0.774), the most sensitive cell line (U-1285) showing least dsb rejoining and the most resistant (U-1810) showing most dsb rejoining of all five cell lines studied. As DNA-PK has been recognised as an important molecular component involved in DNA-dsb repair, we analysed content and activity of this kinase. We found that DNA-PK content and activity correlated with RS (r2 = 0.941 and r2 = 0.944, respectively). The lowest DNA-dependent content/activity was found in the most radiosensitive cells, U-1285 and H-69, whilst the highest content/activity was found in the most radioresistant cells U-1810. These results suggest a correlation between RS and DNA-PK content/activity in lung carcinoma cell lines.

Cells of a small cell lung carcinoma cell line, U-1285, and an undifferentiated large cell lung carcinoma cell line, U-1810, differ in radiosensitivity in parallel to the clinical radiosensitivity of the kind of tumors from which they are derived. The surviving fraction at 2 Gy (SF2) was 0.25 that of U-1285 cells and 0.88 that of U-1810 cells. We investigated the induction of DNA double-strand breaks (DSBs) by X rays and DSB rejoining in these cell lines. To estimate the number of DSBs we used a model adapted for pulsed-field gel electrophoresis (PFGE). The induction levels were of the same magnitude (0.46 and 0.51 DSB/100 Mbp/Gy for U-1810 and U-1285 cells, respectively). These levels of induction do not correlate with radiosensitivity as measured by cell survival assays. Rejoining of DSBs after doses in the range of 0-50 Gy was followed for 0, 15, 30, 60 and 120 min. We found a difference in the velocity of repair during the first hour after irradiation which is parallel to the differences in radiosensitivity. Thus U-1810 cells exhibit a fast component of repair, with about half of the DSBs being rejoined during the first 15 min, whereas U-1285 cells lack such a fast component, with only about 5% of the DSBs being rejoined after the same time. In addition there was a numerical albeit not statistical difference at 120 min, with more residual DSBs in the U-1285 cells compared to the U-1810 cells.

Peptichemio is a complex of six peptides containing m-L-sarcolysin which is used in cancer chemotherapy. One of the peptides, L-propyl-m-sarcolysyl-L-p-fluorophenylalanine (PSF), is highly toxic to melanoma cells. We have compared the effects of melphalan, m-L-sarcolysin and PSF on human melanoma cell lines. PSF was 35-fold and 28-fold more toxic to RPMI 8322 melanoma cells than melphalan and m-L-sarcolysin, respectively. Similar differences in the cytotoxic effects of PSF and m-L-sarcolysin were also seen with two other melanoma cell lines. The cytotoxicity of both PSF and m-L-sarcolysin to RPMI 8322 melanoma cells was potentiated by depletion of cellular glutathione. Both PSF and m-L-sarcolysin caused a protracted induction of DNA cross-links in RPMI 8322 cells, with maximum at 24 hours after drug exposure. PSF induced 9-fold higher levels of DNA interstrand cross-links than m-L-sarcolysin, indicating that the increased cytotoxicity of this drug is associated with a more efficient induction of DNA damage.

Alteration of the melphalan molecule by shifting the di(2-choroethyl)aminogroup from the para- to the meta-position of the phenylalanine residue results in m-L-sarcolysin. By covalent conjugation of different amino acids at the amino- and carboxylgroups of this molecule, a mixture of six peptides known as Peptichemio has been synthesized. In a previous investigation we found that Peptichemio was less toxic to human lymphoblasts than m-L-sarcolysin. In contrast, in the present investigation we found that Peptichemio has higher cytotoxic effect than m-L-sarcolysin on two human melanoma cell lines. The higher cytotoxicity was paralleled by a higher induction of DNA cross-links by Peptichemio as compared to m-L-sarcolysin. A comparative analysis of the six peptides on Peptichemio showed differences in cytotoxic effects on a melanoma cell line. One of the six peptides displayed a considerably higher cytotoxicity than peptichemio itself.

Melphalan inhibits the incorporation of 3H-thymidine and 3H-uridine significantly more in phytohaemagglutinin (PHA)-stimulated human lymphocytes than in a human melanoma cell line (RPMI 8322). Melphalan - induced total DNA cross-linking was 1.7 times higher and DNA interstrand cross-linking was 1.8 times higher in the PHA-stimulated lymphocytes than in the melanoma cells. A higher level of DNA cross-linking was required in melanoma cells than in PHA-stimulated lymphocytes to obtain similar levels of inhibition of incorporation of 3H-thymidine and 3H-uridine. The outflow of cells from G1 to S phase was significantly more inhibited by melphalan in the lymphocytes than in the melanoma cells. Thus the melanoma cells can replicate and transcribe DNA in the presence of levels of DNA damage, which in PHA-stimulated lymphocytes strongly inhibit DNA and RNA synthesis.

Unscheduled DNA synthesis (UDS) indicated by melphalan was studied in freshly collected tumor cells from human melanoma metastases. Comparative studies were done on human bone marrow blast cells. Significant levels of UDS comparable with those in myeloblasts were found in only two of eight melanoma cell populations. This difference between melanoma and blast cells was not related to different cellular uptake of melphalan. When UDS was induced by ultraviolet irradiation, significant levels of UDS were found in all melanoma and blast cell populations studied. Also, in a human melanoma cell line, high levels of UDS were found after exposure to ultraviolet irradiation, while treatment with melphalan did not result in detectable levels of UDS. Possible explanations for the divergent results of UDS in melphalan-exposed melanoma cells are discussed.

Flow cytofluorometric analyses of DNA content were performed on 38 subcutaneous and lymph node metastases from 32 patients with malignant melanoma. Cells from 8 tumors had DNA patterns corresponding to diploid DNA values, and the remaining 30 had aneuploid cell populations, mostly in the diploid to tetraploid region. Eight tumors contained more than one cell population. The number of S-phase cells varied from 3% to 34%. The number of S-phase cells was significantly higher in aneuploid as compared to diploid cell populations. Patients with a proportion of S-phase cells greater than or equal to 10% had a significantly shorter survival time as compared to patients with less than 10% S-phase cells.

Melphalan-induced DNA cross-linking was compared in a human melanoma cell line (RPMI 8322) and in phytohaemagglutinin (PHA)-stimulated lymphocytes. In both cell types a delayed induction of DNA cross-links was observed, with maximum DNA cross-linking occurring 6-12 hours after drug exposure. Significantly higher peak levels of DNA cross-links were found in PHA-stimulated lymphocytes, total DNA cross-linking being 2.5 times and DNA interstrand cross-linking 2.2 times higher. The intracellular content of free melphalan was 1.3-fold higher in RPMI 8322 cells, thus the lower DNA cross-linking was not due to a lower drug concentration in these cells. RPMI 8322 cells had a 1.8-fold higher level of glutathione, possibly indicating a higher capacity of these cells to inactivate melphalan.

DNA flow cytofluorometry of bone marrow cells from 29 patients with multiple myeloma disclosed a 55% incidence of aneuploid cell populations with a DNA index between the diploid and the tetraploid regions (2-4 C) and a mean value of 2.5 C. The bone marrow of these patients demonstrated a correlation between the proportion of aneuploid and morphologically identifiable myeloma cells, which indicates a monoclonal pattern in the aneuploid cases without additional diploid myeloma cell clones. In the present patient material no statistically significant relation was found between diploid and aneuploid cases as regards the type of monoclonal immunoglobulin. Further, there was no difference between the diploid and aneuploid cases as regards myeloma cell differentiation, myeloma cell [3H]-thymidine labeling index or response to melphalan treatment.

The UV-induced DNA repair synthesis in peripheral leukocytes from 7 patients with the nevoid basal cell carcinoma syndrome was compared to that in peripheral leukocytes from 5 patients with basal cell carcinomas and 39 healthy subjects. A dose response curve was established for each individual, and maximum DNA repair synthesis was used as a measure of the capacity for DNA repair. The patients with the nevoid basal cell carcinoma syndrome had about 25% lower level of maximum DNA repair synthesis as compared to the patients with basal cell carcinomas and control individuals. The possibility that DNA repair mechanisms may be involved in the etiology to the nevoid basal cell carcinoma syndrome is discussed.