Hormone and cytokine circadian alteration in non-small cell lung cancer patients.

Alterations in hormone secretion and cytokine levels have been evidenced in many neoplastic diseases. In this study we have evaluated the circadian profile of growth hormone (GH), insulin-like growth factor-1 (IGF-1), interleukin-2 (IL2), melatonin (MEL) and cortisol (COR) serum levels in non-small cell lung cancer patients. Blood was sampled every 4 h for 24 h in 11 healthy (H) men (ages 35-53 years) and 9 men with stage 2, 3 or 4 non-small cell lung cancer (C) (ages 43-63 years). Serum GH, total IGF1, IL2, MEL and COR were measured and examined for group differences, trends, and rhythm characteristics. 24-h means were significantly higher in C234 vs H for GH, GH/IGF1, IL2 and COR, and lower for IGF1, but IL2 and COR were not different for C23 vs H. A linear regression across 4 groups (H, C2, C3, C4) found a positive trend for COR, GH, GH/IGF1 and IL2, and a negative trend for IGF1. A linear regression run between the 24-h mean levels of GH, IGF1, COR, MEL and IL2 in healthy subjects evidenced a statistically significant positive trend between MEL and GH (R = 0.281, p = 0.022) and in cancer patients showed a statistically significant negative trend between GH and IGF1 (R = 0.332, p = 0.01), COR and IGF1 (R=0.430, p=0.001), and a statistically significant positive trend between the 24-h mean of COR and GH (R = 0.304, p = 0.02). Rhythms in MEL and COR (peaks near 01:00h and 08:00h, respectively) indicated identical synchronization to the light-dark cycle for both groups. A circadian rhythm was detected in GH and GH/IGF1 for C23 and H, with IGF1 and IL2 non-rhythmic in any group. In conclusion, an increasing trend and progressive loss of circadian rhythmicity in GH and GH/IGF1, an increasing trend in cortisol and IL2, and a decreasing trend in IGF1 in C, reflect a complex chain of events that could be involved in progression of neoplastic disease. A therapeutic strategy needs to take into account circadian patterns and complex interactions of the multiple functions that characterize the hormone and cytokine levels in the frame cancer progression.

Expression and heterodimer-binding activity of Ku70 and Ku80 in human non-melanoma skin cancer.

BACKGROUND: Experimental data suggest that exposure to ultraviolet radiation may indirectly induce DNA double-strand breaks. AIM: To investigate the contribution of the non-homologous end-joining repair pathway in basal and squamous cell carcinomas. METHODS: Levels of Ku70 and Ku80 proteins were determined by immunohistochemical analysis and Ku70-Ku80 heterodimer-binding activity by electrophoretic mobility shift assay. Matched pathological normal margins and skin from healthy people were used as controls. RESULTS: A significant increase in Ku70 and Ku80 protein levels was found for both tumour types as compared with normal skin (p<0.001). Squamous cell carcinoma showed increased immunostaining as compared with basal cell tumours (p<0.02). A direct correlation was found between Ku70 and Ku80 protein levels and expression of the proliferation markers Ki-67/MIB-1 (p<0.02 and p<0.002, respectively) in basal cell carcinoma. DNA binding activity was increased in basal cell carcinoma samples as compared with matched skin histopathologically negative for cancer (p<0.006). In squamous cell carcinomas, however, the difference was significant only with normal skin (p<0.02) and not with matched pathologically normal margins. CONCLUSIONS: Overall, an up regulation of the Ku70 and Ku80 protein levels seems to correlate only with tumour proliferation rate. As non-homologous end joining is an error-prone mechanism, its up regulation may ultimately increase genomic instability, contributing to tumour progression.

Antibodies elicited by naked DNA vaccination against the complementary-determining region 3 hypervariable region of immunoglobulin heavy chain idiotypic determinants of B-lymphoproliferative disorders specifically react with patients' tumor cells.

Several reports have suggested that the mechanism of protection induced by antiidiotypic vaccination against low-grade lymphoproliferative disorders is likely to be antibody mediated. Here we test the hypothesis that DNA vaccination with the short peptide encompassing the complementary-determining region 3 hypervariable region of immunoglobulin heavy chain (VH-CDR3) may elicit a specific antibody immune response able to recognize the native antigens in the form required for therapy. As a test system, we used the VH-CDR3 sequences derived from two patients with non-Hodgkin's B lymphomas (PA, AS) and one patient with hairy cell leukemia (BA) to immunize outbred Swiss mice. This experimental model could mimic a clinical setting in which different patients present distinct HLA haplotypes. Individual tumor-specific VH-CDR3 sequences were amplified by a two-step procedure and directly cloned into multigenic plasmid vectors (pRC100 and derived) with and without mouse interleukin 2 (mIL-2). Each tumor-specific sequence was characterized by sequencing. Female Swiss mice were vaccinated i.m. with plasmids expressing the tumor-specific VH-CDR3 sequence alone (pRC101-PA), mIL-2 plus the VH-CDR3 sequence (pRC111-PA), or a different unrelated antigen (NS3 of hepatitis C virus; pRC112), the sole mIL-2 (pRC110), and the empty plasmid (pRC100). Boost injections were performed at 3 and 16 weeks from the first vaccination, and sera were drawn before each vaccination and at 6, 9, and 19 weeks. Induction of anti-VH-CDR3s antibodies in the sera and their ability to recognize native antigens on patients' tumor cells were evaluated by FACS analysis. Up to 56% (n = 25) of mice vaccinated with pRC111-PA plasmid and 20% (n = 15) of mice vaccinated with pRC101-PA developed a specific immune response that was maintained throughout 19 weeks of observation in 40% of pRC111-PA-vaccinated mice. No response was detected in sera obtained from mice vaccinated with the other plasmids (n = 45). pRC111-PA injection s.c. was less effective (13%, n = 15) than i.m. injection (53%, n = 15). Indeed, we demonstrated that antibodies elicited by naked DNA vaccination against three different patient-derived VH-CDR3 peptides (pRC111-PA or BA or AS) readily reacted with binding epitopes on the idiotypic proteins expressed on the surface of tumor cells derived from each patient; 60, 40, and 40% of, respectively, PA-, BA-, and AS-vaccinated mice developed specific antibodies. No cross-reactivity was detected among the three different CDR3s against tumor cells derived from the other two patients. The outbred mouse strategy confirmed the significant matching potential of three different VH-CDR3 peptides to be efficaciously presented through different MHCs. We conclude that individual VH-CDR3 DNA vaccination can result in a potentially effective specific immune response against non-Hodgkin's B lymphoma cells by a rapid and low-cost therapeutic approach.

4-Hydroxynonenal, a product of cellular lipid peroxidation, which modulates c-myc and globin gene expression in K562 erythroleukemic cells.

Several studies point to the existence of an inverse correlation between cellular lipid peroxidation and both cell proliferation and neoplastic transformation. Here, we show that 4-hydroxynonenal (HNE) concentrations close to the level found in normal cells (in the range of 1 and 3 microM) can specifically induce changes in the expression of c-myc and gamma-globin mRNA in K562 cells, without inducing any toxic effects or affecting cell viability. Since we have determined that K562 cells have undetectable levels of endogenous lipid peroxidation, all these effects can be assigned to the exogenous HNE treatment. After a 1-h treatment with 1 microM HNE, c-myc mRNA levels decrease transiently during the first 4 h, rebounding later to higher levels, and normalizing to basal expression after 4 days. Run-on experiments show a transient transcriptional block 20 min after HNE treatment and subsequent posttranscriptional regulation. According to S1 mapping, mRNA changes are exerted on c-myc transcripts initiated from both the principal constitutive start sites (P1 and P2). gamma-Globin mRNA levels concomitantly increase 3- to 4-fold, but no significant changes of housekeeping gene expression are observed. On the basis of these results it appears that the restoration in human erythroleukemic K562 cells of HNE concentrations closer to the level in normal cells can modulate the expression of specific genes.

Identification of a mononucleotide repeat as a major target for mitochondrial DNA alterations in human tumors.

Mitochondrial DNA (mtDNA) mutations scattered through coding and noncoding regions have been reported in cancer. The mechanisms that generate such mutations and the importance of mtDNA mutations in tumor development are still not clear. Here we present the identification of a specific and highly polymorphic homopolymeric C stretch (D310), located within the displacement (D) loop, as a mutational hotspot in primary tumors. Twenty-two % of the 247 primary tumors analyzed harbored somatic deletions/insertions at this mononucleotide repeat. Moreover, these alterations were also present in head and neck preneoplastic lesions. We further characterized the D310 variants that appeared in the lung and head and neck tumors. Most of the somatic alterations found in tumors showed deletion/insertions of 1- or 2-bp generating D310 variants identical to constitutive polymorphisms described previously. Sequencing analysis of individual clones from lymphocytes revealed that patients with D310 mutations in the tumors had statistically significant higher levels of D310 heteroplasmy (more than one length variant) in the lymphocyte mtDNA as compared with the patients without D310 mutations in the tumor mtDNA. On the basis of our observations, we propose a model in which D310 alterations are already present in normal cells and achieve homoplasmy in the tumor through a restriction/amplification event attributable to random genetic drift and clonal expansion.

Detection of mitochondrial DNA mutations in primary breast cancer and fine-needle aspirates.

To determine the frequency and distribution of mitochondrial DNA mutations in breast cancer, 18 primary breast tumors were analyzed by direct sequencing. Twelve somatic mutations not present in matched lymphocytes and normal breast tissues were detected in 11 of the tumors screened (61%). Of these mutations, five (42%) were deletions or insertions in a homopolymeric C-stretch between nucleotides 303-315 (D310) within the D-loop. The remaining seven mutations (58%) were single-base substitutions in the coding (ND1, ND4, ND5, and cytochrome b genes) or noncoding regions (D-loop) of the mitochondrial genome. In three cases (25%), the mutations detected in coding regions led to amino acid substitutions in the protein sequence. We then screened an additional 46 primary breast tumors with a rapid PCR-based assay to identify poly-C alterations in D310, and we found seven more cancers with alterations. Using D310 mutations as clonal marker, we detected identical changes in five of five matched fine-needle aspirates and in four of four metastases-positive lymph nodes. The high frequency of D310 alterations in primary breast cancer combined with the high sensitivity of the PCR-based assays provides a new molecular tool for cancer detection.

A somatic mutation in the 5'UTR of BRCA1 gene in sporadic breast cancer causes down-modulation of translation efficiency.

Mutations in the 5' UTR which cause increment/decrement of translation efficiency have been recently described as a novel molecular mechanism of disease. Alterations in the consensus sequence for the translation initiation may promote context-dependent leaky scanning of ribosomes and/or initiation from a downstream AUG codon. Initiation of translation from a downstream in-frame AUG codon in BRCA1 gene was recently identified in normal cells and possibly in breast cancer. Here we present further insight into BRCA1 translational pathophysiology investigating the role of the canonical structure of the initiation consensus sequence of BRCA1. We have analysed the effect of a somatic point mutation (117 G>C) in position -3 with respect to the AUG of the BRCA1 gene, identified in a highly aggressive sporadic breast cancer. We constructed chimeric genes encoding the luciferase reporter sequence downstream of the wild type or the mutated BRCA1 5'UTR. These transcripts were tested for their activity in in vitro and in vivo systems. In in vitro transcription/translation assays the estimated translation efficiency of the construct with the mutated BRCA1 5'UTR was 30-50% lower than that with the wild type BRCA1 5'UTR. The same chimeric genes were analysed for their expression in vivo by transient transfection in human cells. While the two constructs were equally transcribed, the plasmid carrying the mutated sequence produced 70% less luciferase activity compared to the wild type sequence. Finally, to obtain a direct evaluation on translational efficiency in vivo, we analysed mRNA translation on translationally active and non-active ribosomes separated from transfected cells. Mutant mRNA was partially localized in subpolysomal particles analytically confirming a polysome recruitment defect. Thus, characterization of BRCA1 5'UTR and translation efficiency seems to provide new insight into BRCA1 role in breast and ovarian cancer pathogenesis.

Tumor specific modulation of KU70/80 DNA binding activity in breast and bladder human tumor biopsies.

The Ku70/80 heterodimer is the regulatory subunit of the DNA-dependent protein kinase (DNA-PK) and its DNA-binding activity mediates DNA double-strand breaks repair. Although Ku80 was recently proposed as a caretaker gene involved in the control of genome integrity, no data are available on Ku70/80 DNA-binding activity in human tumors. Heterodimer DNA-binding activity and protein expression were assayed by electrophoretic-mobility-shift-assay (EMSA) and Western blot analysis, in nuclear and cytoplasmic extracts from eight breast, seven bladder primary tumors and three metastatic nodes from breast cancers. Corresponding normal tissues of the same patients were used as controls. Ten out of 15 tumors showed nuclear Ku-binding activity 3-10 times higher than in the normal tissues, irrespective of bladder or breast origin. Conversely, in 5/15 primary tumors and in all the metastatic nodes analysed, nuclear Ku-activity was 1.5-4.5-fold lower than in the corresponding normal tissues. Cytoplasmic heterodimer activity significantly differed between tumor and normal tissues, displaying a 2-10-fold increase in neoplastic tissues. Three different patterns combining both Ku expression and activity with tumor characteristics were identified. In low aggressive breast tumors p70/p80 proteins were expressed in tumor but not in normal tissues. The heterodimer binding-activity matched the protein levels. In non-invasive bladder carcinomas no significant differences in protein expression between tumor and the corresponding normal tissues were found, however heterodimer binding-activity was increased in tumor samples. In breast and bladder tumors, at the advanced stage and in node metastases, the binding activity was strongly reduced in tumor biopsies, however no differences were demonstrated between normal and tumor protein levels. Our results suggest a different modulation of Ku70/80 DNA-binding activity in human neoplastic tissues, possibly related to tumor progression. Findings provide further data on tissue-specific protein expression and post-translational regulation of heterodimer activity.

Control of neoplastic cell proliferation and differentiation by restoration of 4-hydroxynonenal physiological concentrations.

Several studies point to the existence of an inverse correlation between cellular lipid peroxidation and both cell proliferation and neoplastic transformation. In anaplastic cell lines products of membrane lipid peroxidation are very low or undetectable. Furthermore numerous results demonstrate effect of lipid peroxidation products on central biochemical pathways and intracellular signalling at physiological concentrations. 4-hydroxynonenal (HNE) is one of the most active products of lipid peroxidation. The restoration of HNE physiological concentrations in neoplastic cells may inhibit cell proliferation and modulate cell re-differentiation. This review try to summarize and critically discuss the effects of physiological concentrations of HNE on normal and neoplastic cell line.

Effect of 4-Hydroxynonenal on cell cycle progression and expression of differentiation-associated antigens in HL-60 cells.

4-Hydroxynonenal (HNE) is a highly reactive aldehyde produced by lipid peroxidation of cellular membranes that inhibits growth and induces differentiation in HL-60 cells. Its mechanisms of action were investigated by analyzing the cell cycle distribution and the appearance of differentiated phenotypes in HL-60 cells. Data obtained by exposing cells to DMSO for 7.5 h (same time as for HNE treatment) or for the whole length of the experiments (5 d) were used for comparison. HNE induced a marked increase in the proportion of G0/G1 cells after 1 and 2 d. The brief DMSO treatment did not affect the distribution, whereas continuous exposure led to a progressive accumulation of cells in G0/G1 (maximal at day 5). The proportion of phagocytic cells gradually increased in HNE-treated and DMSO long-exposed cultures from day 2 and peaked at day 5 (35 and 63%, respectively), whereas the effect of the brief DMSO treatment was negligible. The expression of CD11b and CD67 increased in cells treated with HNE or continuously exposed to DMSO, whereas CD36 was expressed at low levels on both treatments. These results indicate that the pathway of the granulocytic differentiation induced by HNE in HL-60 cells differs from that of DMSO: with HNE, growth inhibition precedes the onset of differentiation, whereas in DMSO-treated cells the two processes are chronologically associated.

The Apolipoprotein E genotype in patients affected by syndromes with focal cortical atrophy.

The role of the Apolipoprotein E (APOE) alleles in syndromes associated with focal cerebral atrophy (fronto-temporal dementia, primary progressive aphasia, corticobasal degeneration) is still controversial. We studied the APOE allele distribution in 39 patients with clinically diagnosed syndromes associated with focal cerebral atrophy (FCA), in 50 patients with early-onset probable Alzheimer's disease (EOAD), and in 60 patients with late-onset probable AD (LOAD). The APOE genotype was determined from a blood sample, using polymerase chain reaction and restriction enzyme digestion. The APOE epsilon4 allele frequency was significantly higher in the EOAD (21.0%) and LOAD (33.3%) groups, but not in the FCA group (5.1%), as compared with controls. In our population, the epsilon2 allele frequency was significantly higher in patients with FCA (12.8%) than in controls (4.8%). These results show that the APOE epsilon4 allele is not a risk factor for syndromes associated with FCA. The potential role of the epsilon2 allele in these syndromes needs further investigation.

Growth inhibition and differentiation induction in murine erythroleukemia cells by 4-hydroxynonenal.

4-Hydroxynonenal (HNE) is one of the major end products of lipid peroxidation. Here we show that the exposure of murine erythroleukemia (MEL) cells to 1 microM HNE, for 10.5 h over 2 days, induces a differentiation comparable with that observed in cells exposed to DMSO for the whole experiment (7 days). The exposure of MEL cells for the same length of time demonstrates a higher degree of differentiation in HNE-treated than in DMSO-treated MEL cells. The protooncogene c-myc is down-modulated early, in HNE-induced MEL cells as well as in DMSO-treated cells. However, ornithine decarboxylase gene expression first increases and then decreases, during the lowering of the proliferation rate. These findings indicate that HNE, at a concentration physiologically found in many normal tissues and in the plasma, induces MEL cell differentiation by modulation of specific gene expression.

Effect of 4-hydroxynonenal, a product of lipid peroxidation, on NK susceptibility of human K562 target cells.

4-Hydroxynonenal (HNE) is one of the major breakdown products generated by lipid peroxidation of cellular membranes. The level of lipid peroxidation and the concentration of its products are inversely related to the rate of cell proliferation and directly related to the level of cell differentiation. It has been reported that HNE inhibits DNA synthesis, ornithine decarboxylase (ODC) activity and c-myc expression in different leukemic cells lines. It has also been demonstrated that HNE inhibits proliferation and induces differentiation in HL60 cell line. In the present study the effects of HNE, at concentrations close to those found in the normal tissues, on the NK susceptibility of human K562 target cells were analyzed. Repeated treatments at 45 minutes intervals with 1 microM HNE were performed to maintain the cells in the presence of the aldehyde for 12 hours. The effect of HNE was compared with that obtained in Haemin-treated cells. HNE causes a strong inhibition of cells growth (53% vs. 34% with Haemin) without affecting cell viability. We further investigated the NK susceptibility of K562 cell line upon in vitro treatment with HNE. Cytotoxic activity of mononuclear cells (MNC) from peripheral blood of healthy donors was determined by 4 hours Cr51-release assay. The results obtained, expressed in terms of percentage of specific lysis at different E:T ratios and in terms of KC (10(6)) at the E:T ratio of 50:1, show that HNE treatment of K562 cells leads to a marked reduction of susceptibility to NK cells; this decrease is very close to that found in the K562 cells treated with Haemin used as inducer. Similar results were obtained using MNC pre-treated with beta-interferon (IFN) as effector cells. MNC show a reduced capacity to lyse HNE-treated cells also under the enhancing cytolytic effect of IFN. These results are in line with data obtained with several common inducers of differentiation such as DMSO, retinoic acid or others.

Effect of 4-hydroxynonenal, a product of lipid peroxidation, on natural cell mediated cytotoxicity.

Lipid peroxidation of cell membrane yields a variety of final products whose a quantitatively important component is 4-hydroxynonenal (HNE). Previous studies performed in our laboratory suggest that HNE may play a physiological role in the control of cellular proliferation and/or differentiation. This appears to be further supported by our recent findings showing that pre-treatment of K562 cells with a physiological concentration of HNE leads to a marked reduction of susceptibility of NK cells. The observed regulatory effects of HNE on tumor cell growth and susceptibility to natural immune resistance, led us to try to better understand the immunotoxicological properties of this aldehyde. The present study analyses the effects of HNE on NK-mediated cytotoxicity. Treatment of MNC as effector cells with concentrations of HNE ranging from 0.001 to 1 microM for 1 h, did not produce noticeable effects on NK activity. Therefore, this aldehyde at physiological concentrations is able to differentiate tumor cells and to down-regulate target susceptibility to NK effectors from one side. On the other side, it is not able to modify the efficiency of the NK function. Moreover, HNE concentrations higher than 1 microM showed significant and concentration-dependent inhibition of NK activity. However, this effect is reversible and can be antagonized, at least in part, by treatment of effector cells with HNE in combination with beta-interferon.

Molecular organization of oncogenes and potentially oncogenic regulatory genes in primary human brain tumors.

We review here results obtained from a wide screening of 72 primary human brain tumors, in order to investigate the molecular organization of proto-oncogenes and potentially oncogenic genes. We demonstrated alterations in the restriction pattern of c-sis oncogene in 1 anaplastic astrocytoma and in 1 endotheliomatous meningioma, and amplifications of c-myc oncogene in 2 endotheliomatous meningiomas. In the same screening of primary brain tumors, we have recently published evidences for a specific alteration in the restriction pattern of estrogen receptor gene sequences which code for the DNA binding domain of the protein. These results, although preliminary and not still sufficient for a conclusive determination of the role in brain tumor growth and/or development, provided evidences of relatively frequent molecular alterations of genes involved in the control of cell proliferation and differentiation.

[Genetic therapy in oncology: ethical aspects]. / Terapia genica in oncologia: aspetti etici.

The more advanced oncologic therapies are directing toward new frontiers, on account of the remarkable undesirable effects of chemio- and radio-therapies. This new therapeutic experiences are of type biological (vaccines), or genic (substitution again genes with shutters meaning-tumoral). This therapies involve, to be effected, some ethical shrewdnesses: choice of the patient, the engineering modality of the genes, the transfer of the genes in cells of the exclusively somatic line, the elimination of the pathogenic risk of the vector virus, the obligatory use of sterile rooms, the attention to the administration of the drug, a legal issue of the judgment of notoriety.

BCR/ABL1 fusion transcripts generated from alternative splicing: implications for future targeted therapies in Ph+ leukaemias.

Philadelphia (Ph+) positive leukaemias are an example of haematological malignant diseases where different chromosomal rearrangements involving both BCR and ABL1 genes generate a variety of chimeric proteins (BCR/ABL1 p210, p190 and p230) which are considered pathological "biomarkers". In addition to these three, there is a variety of fusion transcripts whose origin may depend either on diverse genetic rearrangement or on alternative/atypical splicing of the main mRNAs or on the occurrence of single-point mutations. Although the therapy of Ph+ leukaemias based on Imatinib represents a triumph of medicine, not all patients benefit from such drug and may show resistance and intolerance. Furthermore, interruption of Imatinib administration is often followed by clinical relapse, suggesting a failure in the eradication of residual leukaemic stem cells. Therefore, while the targeted therapy is searching for new and implemented pharmacological inhibitors covering all the possible mutations in the kinase domain, there is urge to identify alternative molecular targets to develop other specific and effective therapeutic approaches. In this review we discuss the importance of recent advances based on the discovery of novel BCR/ABL1 variants and their potential role as new targets/biomarkers of Ph+ leukaemias in the light of the current therapeutic trends. The limits of the pharmacological inhibitors used for treating the disease can be overcome by considering other targets than the kinase enzyme. Our evaluations highlight the potential of alternative perspectives in the therapy of Ph+ leukaemias.