Guy Riou

Association between poor prognosis in early-stage invasive cervical carcinomas and non-detection of HPV DNA

Human papilloma virus (HPV) DNA sequences (HPV types 16, 18, 33, 35 or uncharacterized) were detected by Southern blot hybridisation and polymerase chain reaction in 84% of 106 early-stage invasive carcinomas of the uterine cervix. Among HPV-positive patients, the risk of overall relapse did not differ with individual HPV types. Compared with HPV-positive patients, those with no detectable HPV DNA had a 2.6 times higher risk of overall relapse (p less than 0.05) and 4.5 times higher risk of distant metastases (p less than 0.01). The 24-month relapse-free survival rate in HPV-positive patients was significantly higher than that in HPV-negative patients (77% vs 40%), and the difference was similar (91% vs 56%) among those who were node-negative. These data indicate that HPV-negative cervical carcinomas may represent a biologically distinct subset of tumours that carry a poorer prognosis than do HPV-positive cancers.

C-myc PROTO-ONCOGENE EXPRESSION AND PROGNOSIS IN EARLY CARCINOMA OF THE UTERINE CERVIX

Expression of the c-myc gene was studied by northern blot and slot blot hybridisation in 72 specimens of stage I or II squamous cell carcinoma of the uterine cervix. In 25 of the 72 tumours c-myc proto-oncogene was overexpressed (ie, at levels 4-20 times higher than in normal tissues). Patients whose tumours showed c-myc overexpression had an eight-fold greater incidence of early relapse than the other patients (p = 0.001). The 18-month relapse-free survival rates were, respectively, 49% and 90% for these two groups of patients.

Sequential modifications of topoisomerase I activity in a camptothecin-resistant cell line established by progressive adaptation

The DNA-topoisomerase I (Topo I) inhibitor, camptothecin (CPT), is a plant alkaloid with an important antitumor activity. In order to investigate the cellular mechanism leading to the development of the resistance to this agent, we have established by progressive adaptation a P388 subline resistant to CPT. After 5 months of continuous drug exposure, the resistance index reached a value of 20 and the resistant cell line, P388CPT0.3, was maintained in the presence of CPT. CPT-induced single strand breaks measured by alkaline elution were found drastically reduced in the resistant cell line. Topo I activity and CPT-induced DNA cleavage were measured on cells at different steps of resistance. We first observed that the Topo I activity was strongly decreased. In addition, the resistant cells recovered the ATP-independent relaxation activity after 3 months of exposure to CPT, but still kept a reduced CPT-induced DNA cleavage. Further evaluations at the final stage of the resistance induction have indicated that cells presented a CPT-resistant form of Topo I. Rearranged Topo I gene on one allele and a reduced Topo I transcription were also observed in resistant cells. The putative role of the rearrangement was discussed. These data show that the resistance mechanism has evolved from a decreased Topo I activity to an altered form of the enzyme, and suggest that multiple mechanisms of Topo I modifications could contribute to CPT resistance.

c-myc, p53 and bcl-2, apoptosis-related genes in infiltrating breast carcinomas: evidence of a link between bcl-2 protein over-expression and a lower risk of metastasis and death in operable patients.

Apoptosis is an important physiological process controlled by multiple genes, including c‐myc, p53 and bcl‐2, and its inhibition may lead to the development of human cancers. In this study, we analyzed expression of the c‐myc gene using Northern blot and of the p53 and bcl‐2 proteins by immuno‐histochemistry in 175 breast tumor specimens obtained from patients with operable breast cancer. We evaluated the relation between expression of these 3 genes and (i) the main usual prognostic factors (tumor size, histo‐prognostic grade, hormone receptors and number of positive nodes); (ii) the risk of death and relapse, taking into account these 4 factors, after a mean period of follow‐up of 9.5 years (SD 2 years). Over‐expression of c‐myc, p53 and bcl‐2 was observed in 35%, 23% and 63% of tumors, respectively. Over‐expression of c‐myc was strongly linked to the number of positive nodes (p = 0.0005). p53 protein expression was associated with both high‐grade (p = 0.0001) and hormone receptor‐negative (p = 0.0001) tumors. In contrast, bcl‐2 protein over‐expression was associated with the main favorable prognostic factors and, more particularly, with hormone receptor‐positive tumors (p = 0.0001). Multivariate analysis, using the Cox model, showed that only 2 factors were independently linked to the risk of death: number of positive nodes, which increased the risk (p = 0.0001), and bcl‐2 protein over‐expression, which decreased the risk (p = 0.008). When bcl‐2 over‐expression was studied in relation to nodal status, hormone receptor status and chemo‐ and hormone therapy, no significant difference was observed between different subgroups of patients. bcl‐2 expression was also associated with a significantly lower risk of distant metastasis (p = 0.04). In conclusion, bcl‐2 expression characterizes a particular phenotype of breast cancer with a favorable prognosis, and it may therefore be used as a marker of long‐term survival. Int. J. Cancer (Pred. Oncol.) 84:562–567, 1999.

Prognostic value of c-myc proto-oncogene overexpression in early invasive carcinoma of the cervix.

The prognostic effect of c-myc oncogene overexpression was assessed in a multivariate analysis of 93 patients with invasive carcinoma of the cervix, stage Ib, IIa, and IIb proximal. The treatment was based on the association of brachytherapy-colpohysterectomy and lymphadenectomy. Analysis of c-myc gene expression was done using Northern and slot blot hybridization techniques. Overexpression of c-myc (ie, levels at least three times the mean observed in normal tissues) was present in 33% of the tumors. The proportion of carcinomas with c-myc overexpression significantly increased with the size of the primary tumor (P = .04). No relationship was found between c-myc overexpression and the other clinical and histologic parameters, including the nodal status. The relative risk of relapse (overall, pelvic failure, distant metastases) was analyzed in a Coxs proportional hazards model. Three factors were significantly related to the risk of overall relapse when the multivariate analysis was performed, namely, th...

c-erbB-2 (HER-2/neu) gene amplification is a better indicator of poor prognosis than protein over-expression in operable breast-cancer patients.

Our aim was to compare the prognostic value of c‐erbB‐2 gene amplification analyzed by Southern blot with that of protein (p185) over‐expression measured by immunohistochemistry in 172 patients with operable breast cancer (BC). Amplification and p185 over‐expression were found in 31 (18%) and 51 (30%) BCs, respectively. All but 1 of the tumors showed both amplification and over‐expression, while 21 (12%) tumors displayed over‐expression without amplification. The risk of death associated with c‐erbB‐2 gene amplification and p185 over‐expression was evaluated by multivariate analysis, taking into account tumor size, histoprognostic grade, hormone receptors and axillary node status. During a mean follow‐up of 9.5 (±2) years, node involvement (p < 0.001), c‐erbB‐2 gene amplification (p = 0.02) and negative hormone receptors (p = 0.02) were found to be independent prognostic indicators of the risk of death. Over‐expression of p185 with no amplification was not correlated with this risk. When the risk of death associated with c‐erbB‐2 amplification was studied according to chemo‐ and hormone therapy, no significant difference was observed between subgroups of subjects. Amplification was also associated (p = 0.02) with the risk of multifocal distant metastases (i.e., metastases detected concomitantly in at least 2 sites) and, thus, with BC aggressiveness. These data show the importance of c‐erbB‐2 gene amplification in predicting the long‐term outcome of patients and in selecting eligible patients for c‐erbB‐2–targeted therapies.

Topoisomerase II-mediated DNA cleavage activity induced by ellipticines on the human tumor cell line N417

Ellipticine derivatives have been shown to induce DNA strand breaks by trapping DNA-topoisomerase II (Topo II) in an intermediary covalent complex between Topo II and DNA which could be related to their cytotoxic effects. We report here that Celiptium and Detalliptinium, two ellipticine derivatives clinically used for their antitumoral properties against breast cancer, exhibit the highest in vitro activity on Topo II DNA cleavage reaction and decatenation among a series of 14 ellipticine derivatives. The in vitro cleavage site specificity in pBR 322 plasmid DNA and in a human c-myc gene inserted in a lambda phage DNA is identical for both ellipticines, but different from m-AMSA, another Topo II related antitumoral agent. Recently, it has been shown that the ellipticine derivative Celiptium presents a strong cytotoxic activity in vitro on different human tumors including small cell lung carcinoma (SCLC). However, the studies that involved Topo II as a target for ellipticine derivatives have been performed only by using animal tumor cell lines. Therefore we have studied the in vivo DNA cleavage activity of Celiptium and Detalliptinium on a human SCLC cell line, NCI N417, comparatively to that obtained with m-AMSA. The respective IC50 on cell growth are 9, 8 and 1 microM for Celiptium, Detalliptinium and m-AMSA, respectively. Using the alkaline elution technique, we have observed that Celiptium and Detalliptinium exhibit a weak cleavage activity on genomic DNA from whole cells. The ellipticines are about 50 times less potent than m-AMSA in inducing DNA strand breaks. Analysis of in vivo c-myc gene cleavage by Southern blot hybridization also demonstrates a lack of activity of the ellipticine derivatives as no gene cleavage could be detected up to 50 microM of the drug. With m-AMSA, c-myc gene cleavage is detected at a concentration of 0.2 microM, which indicates that this methodology is less sensitive in detecting DNA strand breaks than is the alkaline elution. Further studies of the drug effect on isolated nuclei by alkaline elution also show that the DNA cleavage activity of Celiptium and Detalliptinium is increased when compared to whole cells. Our data indicate that these two drugs have a weaker cytotoxic effect than m-AMSA on NCI N417 cell line, due to a limited access to the cell nucleus rather than to a lack of activity on Topo II as assessed by in vitro and isolated nuclei experiments.

Characterization of Phytomonas sp. kinetoplast DNA: A plant pathogenic trypanosomal species

Phytomonas sp. which belongs to the Trypanosomatidae family, is a pathogen of plants. Its kinetoplast DNA consists of a huge network of about 7000 catenated minicircles of 2880 ± 30 base pairs each. It represents 30–33% of the total cellular DNA. An AT composition of 65% has been evaluated from the buoyant density xxx = 1.694 . The restriction endonuclease HpaII was the only one able to cleave the minicircles into a single fragment. The other enzymes so far tested (EcoRI, HindIII, HaeIII, BamHI) do not cleave these minicircles significantly. Analysis of the kDNA by Southern blot hybridization shows that minicircles of Phytomonas sp. have little sequence homology with minicircles of other species of trypanosomes. Maxicircles, present in low proportions, have been identified. Our data indicate that the kinetoplast DNA of Phytomonas sp. presents biochemical characteristics which could be used to identify the Phytomonas genus.

Kinetoplast DNA permits characterization of pathogenic plant trypanosomes of economic importance

Summary— Twelve Phytomonas isolates were obtained from different plants originating from several countries and cultured in vitro in complex media. The kinetoplast DNA (kDNA) was purified and observed by electron microscopy. The structure of kDNA from all isolates appeared as a large network of interlocked minicircles with some maxicircles extruding from the network, as has often been shown for Trypanosomatidae. Topoisomerase II resolved the kDNA network into free minicircles which were then analyzed by electron microscopy and by electrophoresis in agarose gel. The minicircle sizes varied from 1.3 to 2.8 kilobase pairs according to the Phytomonas isolate. The analysis by restriction endonucleases revealed a base sequence heterogeneity in the minicircles of 10 of these Phytomonas isolates. By contrast, in 2 Phytomonas isolates, more than 90% of their minicircle content was found to be homogeneous. Most interestingly, the minicircle cleavage patterns were found to be different between Phytomonas isolates and thus could be used to distinguish them.

Comparative study of kinetoplast DNA in culture, blood and intracellular forms of Trypanosoma cruzi

A comparative study has been made on the kinetoplast DNA (kDNA) from I in culture epimastigote, blood trypomastigote and intracellular amastigote stages of Trypanosoma cruzi. The basic properties of the kDNA in all 3 forms were identical. Thus the DNA was in the form of networks of density 1.698-9 g/cm3 and with sedimentation coefficients (S20w) of approximately 5500, the networks being composed of large complexes of minicircular and apparently linear molecules, the former having contour lengths of 0.45 MICROMETer. Several differences were noted. The ultrastructural arrangement of the kDNA in the kinetoplast of the blood stage consisted of three to four double rows of DNA as compared to one double layered row in the other two stages. There was proportionately more kDNA in the blood stages, suggesting that, since the networks have apparently the same size (see above), more than one is present. DNA loops situated at the periphery of the kDNA networks were observed in higher proportion in blood and intracellular forms. Dimeric and oligomeric circles were present in the kDNA of the blood and intracellular stages in much greater proportion than in culture epimastigote stages. Few large circular molecules, heterogeneous in size, were also observed in intracellular blood stages. There were some differences, mainly quantitative, in the gel electrophoresis patterns after endonuclease digestion.