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Despite the best and most aggressive, often integrated, standard therapeutic approaches for mesothelioma, overall survival remains very poor. The actual failure points out clearly the need for the development of novel therapy. One of the promising paths of experimentation is artificial induction of apoptosis. A therapeutic strategy that relies on the down-regulation of BCL-XL inhibition nuclear factor kappaB (NF-kappaB) with a combination of SN38 and tumor necrosis factor (TNF) was studied in human mesothelioma cell lines (MSTO-221H, IST-MES1, IST-MES2, MPP89, H28, H513, H2052, and H290).Cell proliferation (clonogenic assay) was inhibited strongly by the combination of TNF and SN38. Examining the persistence of the NF-kappaB complexes using an electrophoretic mobility-shift assay, it appeared that they still were present at 24 hours in TNF-treated cells. In SN38-treated cells, NF-kappaB complexes persisted for 6 hours. In cells that were treated with combined SN38 and TNF, NF-kappaB complexes disappeared quickly and became undetectable at 6 hours. In flow cytometry analysis, only cells that were treated with combined SN38 and TNF demonstrated significant cellular accumulation in the sub-G0-G1 phase, suggesting a specific induction of apoptosis. Morphologic examination (4,6-diamidino-2-phenylindole staining and electron microscopy) and internucleosomal DNA fragmentation (gel ladder) confirmed rigorously the induction of apoptosis.Because of NF-kappaB inhibition with the combination of SN38 and TNF, the expression of BCL-XL (both the protein [Western blot analysis] and the mRNA [reverse transcriptase-polymerase chain reaction analysis]) was down-regulated, cytochrome c was released into the cytoplasm, caspase 3 was activated (Western blot analysis), and, consequently, apoptosis was triggered. The authors hope that the results of the current study may contribute to the design and implementation of a novel therapeutic approach that improves patients' responses to treatment for mesothelioma.

The demonstration that cell-free circulating DNA detected in the plasma of cancer patients is genetically identical to that of the primary tumor has generated substantial interest, leading to >200 publications (http://www.ncbi.nlm.nih.gov). Recently, Wang et al. (1) reported in this journal that the method chosen for DNA isolation might contribute significantly to mutation detection (in their case, K-ras mutations in the plasma of patients with colorectal cancer). Briefly, they recommended the use of a modified guanidine/Promega resin method (G/R) to isolate DNA, affirming that this method enhances assay sensitivity. We used the same approach to detect K-ras mutations in the plasma of patients with non-small cell lung cancer (NSCLC) and compared the Qiagen vs the G/R method for isolation of circulating DNA. We purified DNA from plasma samples and cancer tissues from 12 patients. The DNA in 2 aliquots of the plasma from each patient was isolated by the …

Hexavalent chromium (Cr[VI]) is classified by the International Agency for Research on Cancer as a group I carcinogen. Although the U.S. Occupational Safety and Health Administration was obliged to reduce the permissible exposure limit (PEL), it was reported that U.S. workers continue to be exposed to dangerously high Cr(VI) levels. In this study, we examined the role of p53 and target genes in a bronchoalveolar carcinoma isogenic cell line system and in primary human bronchial epithelial cells. p53-Negative parental H358 cell line, the same line in which the wild-type p53 expression vector (pC53-SN3) was introduced, and cells obtained from biopsies of human bronchus were exposed to chromate. Induction of DNA strand breaks were evaluated by alkaline elution assay, and apoptosis was analyzed by gel ladder, annexin V-PI staining, and ELISA, whereas p53 and target genes were evaluated by Western blots. Although Cr(VI) induced DNA strand breaks in both H358 cell clones, apoptosis was present only in the p53-transfected cells (H358p53(+/+)). In these cells, Cr(VI)-induced apoptosis is mediated by p53 upregulation of p53-upregulated modulator of apoptosis (PUMA), BAX translocation to mitochondria, cytochrome c release, and caspase-3 activation. In primary human bronchial epithelial cells expressing functional p53, Cr(VI) induced expression of PUMA and Noxa, which promote apoptosis through BAX. This result establishes p53 as the "necessary" player in Cr(VI)-induced apoptosis. To the best of our knowledge, this is the first report indicating strict correlation of Cr(VI) apoptosis to PUMA induction on primary human bronchoalveolar cells in short-term cultures.

Acetylcholine (Ach), one of the most important examples of a neurotransmitter, represents a phylogenetically old molecule, widely distributed from bacteria to humans. The finding that neuronal Ach receptors (nAChRs) are present in non-neuronal cells raised some interesting issues related to their specific activity. In humans, different studies have showed that many lung cancer cells expressed nAchRs and that low concentrations of nicotine blocked the induction of apoptosis in these cells. A recent study presents data that SCLC express a cholinergic autocrine loop that can regulate cell growth. Such work demonstrates that SCLC cells have a cholinergic phenotype and that ACh exerts as an autocrine growth factor in human lung tumors. Recently it has been shown that human malignant pleural mesothelioma express a cholinergic system, involved in cell growth regulation. Hence, mesothelioma cell growth as well as normal mesothelial cells growth is modulated by the cholinergic system in which agonists (i.e. nicotine) has a proliferative effect and antagonists (i.e. curare) has an inhibitory effect. Furthermore apoptosis mechanisms in mesothelioma cells are under the control of the cholinergic system (nicotine antiapoptotic via induction of NF-kappaB complexes and phosphorilation of Bad at Serine(112), curare proapoptotic via G(0)-G(1) arrest p21(waf-1)-dependent, but p53-independent). The involvement of the non-neuronal cholinergic system in lung cancer and mesothelioma appears reasonable and open up new therapeutic strategies.

Aims A case-control study was performed to investigate the relationship between cervical cancer and TP53 polymorphism at codon 72 in young black African women from The Gambia. Materials and Methods The TP53 polymorphism at codon 72 was examined by PCR amplification and SSCP analysis in 40 patients with primary cervical cancer and in 20 healthy women of the same age and from the same geographical area. The occurrence of TP53 polymorphism in combination with the HPV-16 E6 genotype (assayed by PCR) was evaluated. Results The distribution of TP53 genotypes in cervical cancer patients and in the control group was not statistically different (p=0.45) and homozygosity for argine at residue 72 was not associated with cervical cancer (odds ratio: 1.24; 95% confidence interval 0.21-9.16). Similarly, a different genotype distribution, cervical cancer and presence of HPV-16 E6 were not observed. Conclusions These results cannot rule out an association between TP53 polymorphism at codon 72, HPV infection and the etiology of cervical cancer in this population sample.

This study presents data suggesting that both human mesothelioma (cell lines and human mesothelioma biopsies) and human normal mesothelial cells express receptors for acetylcholine and that stimulation of these receptors by nicotine prompted cell growth via activation of nicotinic cholinergic receptors. Thus, these data demonstrate that: (a) human mesothelioma cells and human biopsies of mesothelioma as well as of normal pleural mesothelial cells express functionally alpha-7 nicotinic acethlycholine receptors, evaluated by alpha-bungarotoxin-FITC binding, receptor binding assay, Western blot, and reverse transcription-PCR; (b) choline acetyltransferase immunostaining is present in mesothelioma cells; (c) mesothelioma cell growth is modulated by the cholinergic system in which agonists (i.e., nicotine) has a proliferative effect, and antagonists (i.e., curare) has an inhibitory effect, evaluated by cell cloning, DNA synthesis and cell cycle; (d) nicotine induces Ca(+2) influx, evaluated by [(45)Ca(2+)] uptake, and consequently activation of mitogen-activated protein kinase pathway (extracellular signal-regulated kinase and p90(RSK) phosphorylation), evaluated by Western blot; and (e) apoptosis mechanisms in mesothelioma cells are under the control of the cholinergic system (nicotine antiapoptotic via induction of nuclear factor-kappaB complexes and phosphorylation of Bad at Ser(112); curare proapoptotic via G(0)-G(1) arrest p21(waf-1) dependent but p53 independent). The involvement of the nonneuronal cholinergic system in mesothelioma appears reasonable and open up new therapeutic strategies.

7from our group have showed that MPM expresses a cholinergic system involved in the regulation of both cell growth and the apoptotic machinery. These data now need to be reconciled to construct preclinical models and to approach clinical research from a translational perspective.

A therapeutic strategy that relies on the use of c- myc antisense in combination with a farnesyltransferase inhibitor, RPR-115135 (C31H29NO4), was studied in human cancer cell lines carrying different mutations (Ras, p53, myc amplification). Cell proliferation was strongly inhibited by the combination and was observed when c- myc oligo (at a concentration that down-regulates c- myc expression) was followed by RPR-115135. Cell cycle analysis demonstrated an accumulation in G-G1 phase and a tendency to apoptosis (not detectable in cells treated with a single agent). Morphological examination and DNA fragmentation assays (filter binding and enzyme-linked immunosorbent assay DNA fragmentation) confirmed the induction of apoptosis. Apoptosis was not p53- and/or p21 waf -1-dependent, and the key effector was caspase activation. The combination induced Bax expression and Bcl-2 inhibition. Down-regulation of c- myc amplification carried out a specific role exclusively when Ras was mutated. Exposure of human proliferating lymphocytes to combination did not result in cytotoxicity, suggesting that mechanisms regulating c- myc gene expression during normal T cell proliferation might not be involved. Because of the high percentage of human tumors overexpressing c-myc mRNA and/or protein and, simultaneously, harboring oncogenic Ras mutants (i.e., colon cancers), interrupting the myc- and Ras-signaling pathway would be one of the major focuses on therapy of these types of tumors.

Surgery is the only method of cure in lung cancer. Seldom its application with radical intent is possible. Despite the efforts aimed at integrating all the therapeutic strategies, the overall outcome of the management of this disease remains disappointing. For this reason, in the last three decades, thousands of preclinical and clinical attempts have been realised in order to investigate any possible way to cure this disease and significant steps forward have been made on the basis of the increasing "molecular knowledge" in the so called "post-genomic era". Particularly the impressive step forward in the biological characterization of cancer as a result of genetic/epige netic multistep process has brought in a multitude of variables with staggering classification potentialities. "Benchside" and "bedside" scientists have assembled in functional teams to move the common efforts "translationally" to bridge basic and clinical research for a mutual synergistic enhancement. This paper represents the effort of a lung cancer focused translational research team made up of molecular biologists, medical oncologists and thoracic surgeons to achieve a comprehensive, but simple, review of the current status of the shift from cytotoxic to molecularly targeted therapy in lung cancer treatment potentially useful in the planning of translational research trials.

A new nonpeptidic farnesyltransferase inhibitor, RPR-115135, in combination with 5-fluorouracil (5-FU) was studied in an isogenic cell line model system consisting of human colon cancer HCT-116 cells. HCT-116 cells were transfected with an empty control pCMV vector and with a dominant-negative mutated p53 transgene (248R/W). We found that, relative to control transfectants, there was a slight tendency for the p53 inactivated cells to be less sensitive to 5-FU after 6 days of continuous treatment. Simultaneous administration of RPR-115135 and 5-FU, at equitoxic concentrations, resulted in an enhancement of 5-FU cytotoxicity, especially in the CMV-2 clone. Growth inhibition could be accounted for on the basis of a specific cell cycle arrest phenotype (G 2 -M arrest in CMV-2 and S arrest in mutated clones), as assayed by flow cytometry. The combination RPR-115135 + 5-FU increases apoptotic events only in the CMV-2 clone.

A new non peptidomimetic farnesyltransferase inhibitor, RPR-115135, was studied in an isogenic cell model system consisting of human colon cancer HCT-116 line. HCT-116 cells were transfected with an empty control pCMV vector or with a dominant-negative mutated p53 transgene to disrupt p53 function. Growth inhibitory effects of RPR-115135 were evaluated on cells growing under different conditions (serum starvation, serum starvation and recovery, nocodazole treatment). The cytotoxic activity of RPR-115135 was independent of the cell cycle status of the target cells. Addition of RPR-115135 only to cells exposed to reduced serum conditions (0.1% FCS) resulted in an enhanced ability of HCT-116 cells to arrest in the G 0 /G 1 phase. This arrest response appeared independent of p53/p21 cipl/waf-1 function. A reduction of Cyclin A protein amount by RPR-115135 was observed in both clones. These latter results suggest that RPR-115135 migth down-regulate the cell cycle factor that would normally impede G 0 /G 1 arrest.

To investigate the relationship between oncogene activation and induction of micronuclei by a new non-peptidic mimetic farnesyltransferase inhibitor, RPR-115135, two isogenic cell lines, human colon cancer line HCT-116, which harbors a K-ras mutation, and spontaneously immortalized human breast epithelial cell line MCF-10A, were utilized. HCT-116 cells were transfected with an empty control pCMV vector (clone CMV-2) or with a dominant negative mutated p53 transgene (clone Mu-p53-2) to disrupt p53 function. In both clones RPR-115135 induced a significant increase in the frequency of micronucleation at concentrations that did not affect cell membrane integrity. RPR-115135 produced a significant increase in the ratio of CREST+ to CREST- micronuclei. MCF-10A cells were stably transfected with either c-Ha-ras or c-erbB-2 or both H-ras + c-erbB-2. No induction of micronuclei was observed. No induction of micronuclei was reported in human lymphocytes and in primary spinal cells obtained from 7-day chick embryos. In conclusion, RPR-115135 acts as an aneugenic agent in a complex manner, dependent upon the complement of mutations in cell regulatory genes in tumour cells and this activity may be independent of ras genotype.

Translational Relevance Recent studies strongly suggest that receptors for nicotine play a significant role in lung cancer predisposition and natural history. These data give a new thrust to research efforts assessing the potential curative (antitumor) value of natural compounds that are well

We describe a simple and fast method for the detection and localization of low copy numbers of HPV DNA in formalin-fixed and paraffin-embedded archival tissues. We have developed a protocol for direct bl situ-PCR in order to demonstrate its convenience in rapid and reproducible assessment of HPV infection in unknown biopsies. The morphological aspect of the tissues has been maintained, despite the multiple steps of fixation, permeabilization and thermal cycling, and positivity has been detected only in virus target cells.

Taxol is an antitumor drug which, as its mechanism of action, promotes microtubule assembly in vitro. Camptothecin (CPT) is an anticancer agent with the peculiar mechanism of poisoning eukaryotic DNA topoisomerase I. Both drugs are in clinical trials and their chemotherapeutic efficacy seems promising in refractory human ovarian cancer. We studied the molecular and cellular pharmacology of the two drugs when administered simultaneously toward human ovarian cancer cell line A2780. Taxol inhibits CPT-induced single-strand breaks as well as CPT-induced cytotoxicity. Taken together, our experiments indicate that the two drugs might interact with the same class of nuclear enzyme, i.e. DNA topoisomerase I.

The effect of Tumor Necrosis Factor (TNF) on VP16 or VM26 cytotoxicity was studied in a human glioblastoma cell line T98G, which expresses TNF-receptors. Although T98G cells did not produce TNF endogenously they were resistant to the cytolytic effect of TNF. T98G cells were also moderately sensitive to the action of VP16 or VM26. TNF given at 1000 U/ml was able to increase the cell drug-sensitivity significantly. This effect was due to an increase in the VP 16-induced cleavable-complexes by TNF. These findings suggest that TNF specifically sensitizes human glioma T98G cells to the effects of VP 16 of VM26.

The effect of interferon-alpha or beta on platinum analogues [cisplatin (CDDP) and carboplatin] cytotoxicity was studied in four glioblastoma cell lines (U373MG, T98G, A172 and U118Mg). All cell lines were strongly resistant to the cytotoxic effect of CDDP or carboplatin. Although both interferons were not cytotoxic in all cell lines, they were able to significantly increase the cell platinum-sensitivity. Specifically interferon-alpha increased the magnitude of CDDP-induced DNA interstrand crosslinks. Our findings suggest that interferons are able to induce a very strong potentiation of platinum analogues cytotoxicity in drug-resistant human glioma cell lines.

We have studied the ability of 8-methoxycaffeine (8-MOC)--one of the most effective caffeine derivatives in inducing chromosomal aberrations--to induce DNA double strand breaks (DSB) in purified human T lymphocytes during the cell cycle. Etoposide- or ellipticine-mediated DNA break frequency was used as a parameter of topoisomerase II activity. DNA-DSB induced by either 8-MOC or VP16 or ellipticine rose co-ordinately with the level of DNA topoisomerase II and with the onset of DNA replication. At concentrations between 10 and 50 microM 8-MOC was approximately 75% as active in terms of DSB as VP16 and ellipticine. By contrast with VP16 and ellipticine, 8-MOC was not cytotoxic. In conclusion, our data suggest that 8-MOC is an agent that efficiently induces DNA-DSB at non-toxic concentrations, and without direct inhibition of topoisomerase II.

A2780 human ovarian cancer cell line and its multidrug resistant counterpart A2780-DX3 were utilized for this in vitro study. A2780-DX3 is resistant in various degrees to several topoisomerase II inhibitors but sensitive to vinca alkaloids. Simultaneous treatment of the A2780-DX3 line with 1000 U/mL rHuTNF largely reverses resistance to most topoisomerase II inhibitors. By itself, 1000 U/mL rHuTNF is not toxic to the resistant line. Uptake and retention of [3H]-Mitoxantrone are not modified by rHuTNF, whereas rHuTNF is very active in potentiating the effects of Mitoxantrone. After treatment with topoisomerase II inhibitors, Doxorubicin, Mitoxantrone, or VP16, rHuTNF restores DNA-SSB and DNA-protein cross-links in the resistant line to the level of the wild type. The cleavage activity of topoisomerase II in the resistant line is about 40% of the level present in the parental line. Five minutes after the addition of 1000 U/mL of rHuTNF, the cleavage activity in the resistant line is about 85% of the level present in the parental line. The catalytic activity of topoisomerase II is only 15% lower in the resistant line, but it is increased by about 50% 5 min after the addition of rHuTNF to the resistant line. These effects are transient and cannot be observed after 30 min. These transient direct effects of rHuTNF on topoisomerase II could be associated with its ability to restore sensitivity to inhibitors of topoisomerase II in the A2780-DX3 line.

The p53 homologue p73 is overexpressed in many tumors, including lung cancer. We have evaluated the differential expression and subcellular localization of the functionally distinct apoptotic (TA) and anti-apoptotic (DeltaN) isoforms of p73 in non-small cell lung cancer (NSCLC), their possible association with p53 expression and determined the methylation status of the two p73 gene promoters (P1 and P2) in this tumor type. Immunohistochemical analysis showed that both isoforms are expressed in the majority of cases. However, the oncogenic DeltaN variant, derived from the transcripts DeltaN'p73 (from P1) and/or DeltaNp73 (from P2), is localized mainly in the nucleus, while the anti-oncogenic TAp73 isoform (derived from a P1 transcript) is sequestered in the cytoplasm in almost all cases analyzed. Significant correlation was found between p53 and DeltaNp73 expression (p=0.041). Methylation analysis conducted on 41 tumor samples showed that the P1 promoter is almost invariably unmethylated (39/41 cases) whereas P2 was found completely methylated in 17 cases and partially or totally unmethylated in 24 samples. No correlation was found between the methylation status of P1 and P2 and p73 expression. Our results demonstrate that both isoforms contribute to p73 overexpression in NSCLC and suggest that their different intracellular localization may reflect an alteration of the functional p53-p73 network that might contribute to lung cancer development.

8-Methoxycaffeine (8-MOC) is a caffeine derivative, more potent than the parent compound, but very similar to caffeine in terms of induction of DNA single-strand breaks (SSBs), DNA double-strand breaks (DSBs) and DNA-protein crosslinks (DPCs). We have studied the capability of 8-MOC, caffeine and 8-chlorocaffeine (8-CC) of inducing SSBs, DSBs and DPCs, and we have compared 8-MOC with ellipticine, a typical inhibitor of DNA topoisomerase II. The DNA effects of 8-MOC appeared similar to those of ellipticine. In both cases SSBs, DSBs and DPCs were present in a similar ratio, and they were rapidly reversible after removal of the drug. The dose-response curve was bell-shaped for both compounds. In addition, 8-MOC, caffeine and 8-CC were capable of inhibiting DSBs induced by ellipticine. These results were obtained at the level of L1210 cell nuclei. In spite of these functional similarities, 8-MOC, caffeine and 8-CC were unable to stimulate the formation of a cleavable complex by purified L1210 topoisomerase II (p170 form) when SV40 DNA and human c-myc DNA were used as substrate. These methylated oxypurines could be active on a different form of topoisomerase II, or, alternatively, they could be active only in the natural chromatin 'milieu' within the nucleus.

A 2780 human ovarian cancer cells, obtained from an untreated patient, have been exposed to a relatively low, clinically maintainable dose (10 nmol/l) of the anthracycline doxorubicin (DX) to derive a low-degree (5-fold) drug-resistant subline (A2780-DX1). Compared to parental cells, these DX-resistant cells have increased size (+60% of cell volume) and contain a greater number of cytoplasmic vacuoles as determined by electron microscopy. When exposed to several other antiproliferative drugs, A2780-DX1 cells were highly cross-resistant (greater than 10-fold) to epirubicin, mafosfamide and cisplatin and slightly cross-resistant (2- to 3-fold) to navelbine and bleomycin, while they retained the original sensitivity to vinblastine, Ara-C and fluorouracil. Gel electrophoresis of cytoplasmic membrane proteins showed differences between the pattern of parental A2780 sensitive and A2780-DX1 cells as far as low-molecular-weight proteins (less than 45 kD) are concerned, while no clear overexpression of P-glycoprotein (P-170) could be detected. Membrane modifications yielding a decrease of both DX uptake and retention, increased content of intracellular glutathione (+32%) and reduced DNA double-strand breaks seem to be involved in the resulting multidrug-resistant phenotype of A2780-DX1 cells.

We appreciated the reading of the work of Amar et al. [1]. The authors reported data suggesting that markers of inflammation such as C-reactive protein (CRP) and IL-6 can help to classify patients who are at high risk for major postoperative complications (PC) following thoracic surgery. Systemic inflammation is thus considered to be important in the pathogenesis of important PC. Defining the precise inflammatory response represents a sensitive issue, frequently debated within scientific community. Plasma levels of cytokines or CRP are probably not sufficient to determine whether a patient or experimental animal is hyper-inflammatory or hypo-inflammatory. If only the pro-inflammatory mediators are measured, then the patient will appear hyper-inflammatory. Conversely, if only cytokine antagonists or anti-inflammatory mediators are measured, the subject appears to be hypo-inflammatory. Indeed, both proand antiinflammatorymediatorsmaybecirculating at the same time in the plasma [2]. Better methods for determining the precise immunological status may be achieved via either a multiplex format for cytokinemeasurements or an evaluation of cellular function. Many cellular aspects become dysfunctional in inflammation, for example in chronic obstructive pulmonary disease (COPD), and may be characterized as either excessive activation or depressed function. Excessive activation refers to cells that are primed so that they respond in a very vigorous manner to a second stimulus (for example, neutrophils generating excess toxic products that cause damage to nearby cells [3]). Conversely, an example of depressed functionwould be neutrophils’ failure to phagocytize any clearly invading pathogens. One of the current areas of active investigation concerning cellular function is the induction of cellular apoptosis or necrosis. The signalling mechanisms and molecules that induce apoptosis are currently being described in great detail by several investigators. The pathobiology of COPD (a disease that can be considered a model of pulmonary inflammation) encompasses multiple injurious processes including inflammation (excessive or inappropriate innate and adaptive immunity), cellular apoptosis, altered cellular and molecular alveolar maintenance program, abnormal cell repair, extracellular matrix destruction (protease and antiprotease imbalance), and oxidative stress (oxidant and antioxidant imbalance) [4]. Apoptosis may contribute to the pathogenesis of sepsis by the delayed removal of those cells that shouldbe removed, i.e., neutrophils, andearly removalof those cells that should not be removed, i.e., lymphocytes. Activated protein C has emerged as a novel therapeutic agent for use in selectedpatientswith severe sepsis, even though the mechanism of its benefit is not well established. It has anticoagulant, anti-inflammatory, antiapoptotic, and profibrinolytic properties, but it is not clear through which of thesemechanismsAPCexerts its benefit in severe sepsis. Focus has recently turned to the role of APC in maintaining endothelial barrier function, and in vitro and in vivo studies have examined this relationship [5]. In conclusion, CRP and IL-6 alterations are extremely nonspecific, and an accurate diagnostic test for sepsis would be a welcome addition in the management of specific patients undergoing thoracic surgery, whereas the condition supporting the surgical indication could easily be correlated with a previous pulmonary inflammation, i.e., lung cancer arising in COPD patients.

The purpose of the work reported here was to investigate, with the alkaline elution technique, the capability of in vivo administered actinomycin D, daunomycin and mitomycin C to induce DNA damage, DNA interstrand cross-linking and DNA-protein cross-linking. The ability of these compounds to induce increases in sister chromatid exchange (SCE) in the bone marrow cells of mice was also investigated. Actinomycin D and daunomycin were active in inducing single strands breaks, while mitomycin C was inactive. Mitomycin C showed a clear DNA interstrand cross-linking activity, while this activity was absent in actinomycin D and daunomycin. All three compounds were positive for SCE induction, but mitomycin C was by far the most active compound. Our results seem to suggest that stabilized intercalating agents are often detectable with the alkaline elution technique, after treatment in vivo. However, they cannot be evaluated with the simple alkaline elution technique only. It is convenient to add to the basic method the modification for detecting cross-links. Finally, DNA interstrand cross-linking and sister chromatid exchanges could be correlated.

For 59 chemical compounds, we have found homogeneous data on transformation in vitro, mutagenicity in the Ames test, and carcinogenicity. We have compared the potency in inducing transformation in vitro in hamster fibroblast cells with the carcinogenic potency and found a modest correlation coefficient between the two parameters (r = 0.37). For these same 59 compounds it was also possible to compare mutagenic potency in the Ames test with carcinogenic potency. The correlation level was very similar (r = 0.34). The predictivity of transformation in vitro increased significantly when only compounds for which some kind of dose-response relationship was available were utilized (r = 0.65). This result stresses the importance of the quantitative aspect of the response in predictivity studies. The present study is compared with previous studies on the quantitative predictivity of different short-term tests. Our work is not definitive, but gives an idea of the possible type of approach to the problem of comparing quantitative predictivities.

Nitrofurantoin was not positive as a carcinogen in long term assays. In vitro it was positive in some short term tests and negative in others. We have examined Nitrofurantoin for its capability of inducing DNA damage in vivo. With the alkaline elution technique, Nitrofurantoin appeared clearly positive in all the tissues examined (liver, kidney, lung, spleen and bone marrow). In the liver we also observed some cross-linking effect. In bone marrow cells Nitrofurantoin was also clearly positive in terms of sister chromatid exchanges (SCEs) induction. DNA damage in vivo was also examined with a viscosimetric method, more sensitive than alkaline elution. With this method the results were essentially negative, suggesting that the two methods detect different types of damage. In view of its positivity in many organs and in two short term tests in vivo, the carcinogenic potential of Nitrofurantoin should be reconsidered.

Induced hyperglycemia in normal subjects increases alpha 2-macroglobulin (alpha 2M) activity and alpha 2M concentration and reduces antithrombin III (ATIII) activity, while it does not affect ATIII plasma concentration. Hyperglycemia-determined variations in ATIII activity and alpha 2M molecules are correlated in an inverse and parallel fashion. A compensatory role for the increase in alpha 2M in the regulation of the coagulation system may be hypothesized. Moreover, these data provide evidence that hyperglycemia may decrease, directly, the biological function of some proteins and may influence the levels of some risk factors for the development of complications in diabetes.

The effects of hyperglycemia on plasma fibrinopeptide A (FPA) levels in normal subjects are reported. An increase of FPA concentration parallel to sustained hyperglycemia was observed; when the glycemia returned to basal values, FPA showed values in normal range. Heparin infusion was able to significantly decrease the hyperglycemia-induced augment of FPA levels. Isovolumic-isotonic NaCl solution infusion produced a slight (NS) increase in FPA levels; however, mild hyperglycemia, achieved by glucagon, was also able to produce a significant increase in FPA concentration. These data demonstrate the direct role of hyperglycemia in conditioning FPA level, and suggest that hyperglycemia, by itself, is a sufficient stimulus to produce thrombin activation in humans.

Excerpt To the editor: A tendency for erythrocytosis among heroin users has been reported (1, 2). Among the factors suggested as contributing to this effect are intermittent respiratory depression ...

To the Editor. —In a recent article, we reported increased levels of glycosylated hemoglobin (Hb A lc ) that demonstrated the existence of an altered glycemic control in addicts similar to that seen in diabetics. 1 Impaired lipid metabolism is a widely recognized phenomenon in diabetics, 2 for this reason, we measured the levels of plasma glucose, Hb A lc , high density lipoprotein-cholesterol, and apolipoprotein A and B in 20 male opiate addicts (aged, 23 ± 3.6 years; weight, 68.4 ± 4.5 kg [mean ± SD]) and in 20 healthy male control subjects who were matched for age (24 ± 3.6 years) and weight (69 ± 3.87 kg). The selected addicts had no humoral signs of liver damage. Confirming the results of our previous study, 1 the concentration of Hb A lc was significantly increased in the addicts (7.1 ± 0.15%) than in the control subjects (6.2 ± 0.18%), P

Increased alpha 2-macroglobulin (alpha 2M) activity and concentration, and decreased antithrombin III (ATIII) plasma concentration are reported in diabetic subjects. In diabetes an inverse correlation between ATIII activity and blood glucose, HbA1, alpha 2M activity and alpha 2M concentration, and a direct correlation between both alpha 2M activity and alpha 2M concentration with blood glucose and HbA1 are found. Moreover, a direct correlation between alpha 2M activity and alpha 2M concentration fails. In both diabetic and normal subjects induced hyperglycemia increases alpha 2M activity and alpha 2M concentration reduces ATIII activity, while ATIII concentration is not affected. These data which show that hyperglycemia may increase alpha 2M molecule levels while altering only the biological function of ATIII, provide evidence that hyperglycemia may decrease, directly, the biological function of some proteins and may condition the levels of some risk factors for the development of diabetic complications such as alpha 2M.

DNA damage induced in vivo by 3′-methyl-4-dimethylaminoazobenzene (3′CH3DAB) was investigated with 2 differently sensitive techniques: the alkaline elution assay and the visometric measurement of DNA damage. 3′CH3DAB appeared to be falsely negative with the alkaline elution assay, whereas with the viscometric approach, which is about 30–50 times more sensitive, it appeared positive, and the DNA damage was dose-dependent.

We have recently observed that different promoting treatments in rat liver carcinogenesis induce an apparent DNA fragmentation, as monitored by the alkaline elution technique (DNA elution through calibrated pores of filters after lysis of nuclei with 2M NaCl) and sedimentation in alkaline sucrose gradients1,2. A summary of the results obtained with 1% orotic acid in the diet of rats for five weeks is shown in Table 1, and a summary of the results obtained with a choline deficient diet for five days is shown in Table 2. Birnboim has reported the induction of DNA fragmentation in leucocytes after treatment with TPA3.