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p53-Related genes, p73 and p63, encode 2 classes of proteins, TA-p73/p63 and DeltaN-p73/p63. TA-p73/p63 demonstrate p53-like properties including gene transactivation and cell death promotion, whereas DeltaN-p73/p63 lack these p53-like functions. Although p53-deficient cancer cells are often less responsive to chemotherapy, they are not completely drug resistant, suggesting that other apoptotic pathways are at work. Here, we compared for the first time to our knowledge p73 and p63 activation in various breast cancer (BC) cell lines after Adriamycin (ADR) treatment, an agent considered as mandatory in breast cancer chemotherapy. Our study was carried out using 1 p53-proficient BC cell line (MCF7 cells) and 3 BC cell lines deficient in p53 response (MCF7/ADR(IGR), MDA-MB157 and T47D) after ADR-induced genotoxic stress. We report that in cells with no p53 response after ADR treatment, TAp73, but not TAp63 or DeltaN-p73/p63, may replace p53 in triggering not only apoptosis but also cell cycle arrest or DNA repair effectors such as p21, GADD45, 14-3-3sigma and p53R2. We also demonstrate that TAp73 siRNA inhibits the accumulation of TAp73 in response to ADR treatment in MDA-MB157 cells and confers protection against ADR. ADR-induced downregulation of the DeltaNp73 isoform in the T47D cell line with nonfunctional mutant p53 further supports anti-apoptotic function of the isoform antagonistic to both p53 and TA-p73/p63. Exogenous TAp73 and DeltaNp73 overexpression in p53-response-deficient cell lines further confirms these results. cDNA microarray techniques demonstrated that the cellular response induced by p73 during ADR treatment could involve specific genes.

Based on gene sequence homologies, a p53 (TP53) gene family become apparent with the addition of the most recently identified p63 (TP73L; formerly TP63) and p73 (TP73) genes to the already known p53. The p53 gene encodes for a unique protein eliciting well-known tumor suppressor gene (TSG) properties that mediate cellular response to DNA damage, e.g., cell cycle arrest or apoptosis. In contrast, both homologues specify an array of isoforms different in their N- and C-terminal domains. Transactivating isoforms, such as TAp63/p73, show TSG properties similar to p53, while isoforms lacking N-terminal transactivating domain such as DeltaNp63/p73, induce a functional block against p53 as well as TAp63/p73 activities. Both p63/p73 types of isoforms are involved in development: p63 is critical for epithelial stem cell renewal and epithelial homeostasis, and p73 is involved in neurogenesis and natural immune response. These facts support interdependent functions for the p53 family members, which appear linked together in a complex and tight regulation network to fulfill cellular functions related to DNA damage and tissue homeostasis maintenance. The lack of p63/p73 mutations in human cancers rule out a typical TSG role for either of the p53 homologues. Nonetheless, p63 and p73 genes seem strongly involved in malignancy acquisition and maintenance process because of: 1) their tissue identities, and 2) their close interplay activities within the p53 family members, and primarily through the negative regulatory role played by DeltaNp63/p73 isoforms for cell death control and differentiation., (Copyright 2003 Wiley-Liss, Inc.)

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., (Copyright 2001 Wiley-Liss, Inc.)

We have studied the occurrence and association of 11q deletions with other chromosomal imbalances in Stage 4 neuroblastomas. To this purpose we have performed comparative genomic hybridization (CGH) analysis on 50 Stage 4 neuroblastomas and these data were analyzed together with those from 33 previously published cases. We observed a high incidence of 11q deletion in Stage 4 neuroblastoma without MYCN amplification (59%) whereas 11q loss was only observed in 15% of neuroblastomas with MYCN-amplification (p = 0.0002) or 11% of cases with 1p deletion detected by CGH (p = 0.0001). In addition, 11q loss showed significant positive correlation with 3p loss (p = 0.0002). Event-free survival was poor and not significantly different for patients with or without 11q deletion. Our study provides further evidence that Stage 4 neuroblastomas with 11q deletions represent a distinct genetic subgroup that typically shows no MYCN-amplification nor 1p deletion. Moreover, it shows that neuroblastomas with 11q deletion also often present 3p deletion. This genetic subgroup shows a similar poor prognosis as MYCN amplified 4 neuroblastomas., (Copyright 2001 Wiley-Liss, Inc.)

Background: Stage IV neuroblastoma is characterized by tumor invasion and metastatic dissemination. Cell lines derived from such neuroblastomas have a high in vitro proliferation capacity., Procedure: We established three neuroblastoma cell lines derived from involved bone marrow of three patients with stage IV neuroblastoma and performed a cytogenetic study., Results: Various culture conditions allowed us to distinguish two cell subpopulations: malignant neuroblasts (Nb-type) and substrate-adherent stromal cells (Str-type). Karyotypic analyses revealed two specific chromosomal abnormalities in diploid malignant IGR-N-331 neuroblasts, der(1)t(1;7)(p22;q11) and der(5)t(5;17)(q35;q21), one unbalanced translocation der(1)t(1;17)(p35;q21)x2 in hyperdiploid malignant IGR-N-337 neuroblasts, and a normal karyotype in both corresponding stromal subpopulations. In contrast, in the IGR-N-91 model, both cell types shared two unbalanced translocations, t(1;4)(q12;p15) and t(2;10)(p14;q11), suggesting that stromal cells and malignant neuroblasts originate from a common stem cell., Conclusions: Based on our findings, we postulate that genetically modified stromal cells may influence the metastatic potential of malignant neuroblasts.

Background and Procedure: The p53 gene homologue, p73, is located on the 1p36-3 locus, which is frequently deleted in human neuroblastoma (NB). A survey of 61 NB showed that among 33% of informative cases, p73 loss of heterozygosity (LOH) occurred in 7 of 20 (35%)., Results: LOH pattern of vicinal markers suggested that the p73 gene could not be considered as the candidate NB suppressor gene. Moreover, comparative measurements of allelic expression in tumors and corresponding patient lymphocytes indicate that pure biallelism is much more frequent in lymphocytes than in tumors (71% vs 30%, P= 0.05), which suggests that disequilibrated allelic expression is associated with NB disease., Conclusion: Therefore, in the p73 LOH NBs, the p73 gene could be altered in the maintained allele not by mutations [Ishimiya et al.: Med Pediatr Oncol, this issue], but rather by an abnormal transcription.

Complete or partial gain of the long arm of chromosome 17 (17q) has been shown recently by molecular cytogenetic techniques to be the most frequent chromosomal change in neuroblastoma and to be associated with adverse prognosis. Few reports, however, have focused on the precise mapping of the commonly overrepresented region. We have investigated 17q gain by the analysis of allelic imbalances at microsatellite loci dispersed along chromosome 17 in a series of 69 neuroblastomas. Allelic imbalances for at least two consecutive loci were observed in 39/59 informative cases, that is in agreement with previously reported frequencies of 17q gain. In a subset of the cases, comparative genomic hybridization analysis established the relationship between these allelic imbalances and the gain of 17q material. A partial 17q gain was observed in 9 cases, delineating a common region of 17q gain between the marker D17S787 (75 cM, 360 cR) and the telomere. In most cases, molecular results were suggestive of partial tri- or tetrasomy, whereas in 4 cases a higher copy number was documented. Our results also confirm that the presence of additional 17q material is closely associated with 1p36 deletion, MYCN amplification, and diploid or tetraploid chromosomal content. Genes Chromosomes Cancer 28:276-284, 2000., (Copyright 2000 Wiley-Liss, Inc.)

An experimental model of advanced human neuroblastoma, IGR-N-91, which is able to disseminate in the nude mouse, has been described. The present study was designed to ascertain which cell population from the IGR-N-91 primary tumour actually disseminates throughout the animals. In s.c. IGR-N-91 tumour xenografts, 3 areas, called pearly, vascularized and haemorrhagic, depending on the presence of blood vessels and haemorrhagic suffusions, were consistently observed and independently resected. Molecular analysis of tumour materials revealed a significant increase in MYCN and max gene transcript levels in the haemorrhagic area, as compared with the pearly and vascularized areas. Given the growth kinetics observed both in vitro and in vivo, and the DNA flow-cytometry profiles of tumour cells obtained from the haemorrhagic area, this transcriptional increase did not appear to be associated with enhanced proliferation. In this area of the tumours, multidrug-resistance-related genes, i.e., MDRI, MRP, GST-pi and topoisomerase II alpha were activated concomitantly with MYCN and max genes. The same observations were made, except for the topoisomerase-II alpha gene, when sub-lines derived from metastases were compared with that derived from the primary tumour. These data demonstrate that over-expression of several genes determining the multi-drug-resistance phenotype precedes the metastatic spread of IGR-N-91 NB tumour cells in the nude mouse. Data also suggest that the cell sub-population exhibiting this pleiotropic over-expression within the primary tumour undergoes selection during metastatic dissemination.

Neuroblastoma shows remarkable heterogeneity, ranging from spontaneous regression to progression toward highly malignant tumors. In search of genetic abnormalities that could explain this variability, we have characterized neuroblastoma tumors by using multiple fluorescent hybridizations. Our results indicate that chromosome 17 is rearranged very frequently in the form of unbalanced translocations with numerous chromosomal partners, all leading to the presence of supernumerary copies of a 25 Mb chromosomal region originating from 17q23.1-qter. Additional 17q material was detected in more than 90% of untreated high-grade neuroblastomas and, along with 1p36 deletion, should represent the most frequent genetic abnormality of neuroblastoma observed until now.

Cisplatin-induced apoptosis and p53 gene status were analyzed in human ovarian carcinoma using a parental IGR-OV1 line and a derived cisplatin-resistant IGR-OV1/DDP subline. Compared with parental cells, cisplatin-resistant cells exhibited a 5-fold higher resistance index and a 2-fold longer doubling time. Cisplatin induced apoptosis in both cell lines, as assessed by cell morphology and the presence of a DNA ladder. However, high concentrations were necessary to induce apoptosis in resistant cells. These cells elicited a 5-fold decrease in the number of platinum atoms bound per nucleotide. IGR-OV1/DDP cells also exhibited enhanced drug efflux and a higher glutathione content. Our data suggest that the levels of cisplatin-DNA lesions are critical for drug sensitivity and apoptosis induction in this in vitro ovarian carcinoma model. Comparative analysis of the p53 gene in sensitive and resistant cells revealed the presence of the same heterozygous mutation in exon 5. A 2-fold increase in p53 mRNA and protein amounts was observed in resistant cells as assessed by Northern and Western blots, respectively. Immunocytochemical staining revealed a higher percentage of p53 stained nuclei in resistant cells. RT-PCR analysis of p53 transcripts showed that both wild-type and mutated alleles were transcribed in sensitive as well as in resistant cells. However, mutated transcripts were 1.5-fold more abundant than wild-type transcripts in sensitive cells, whereas they were 2-fold higher in resistant cells. In addition, mdm-2 protein was over-expressed in resistant cells. Our results address the question of the functionality of p53 protein and its possible role in apoptosis induction in this model. In resistant cells, p53 protein might be inactivated by 2 mechanisms: mutation and complexation with mdm-2 protein. Therefore, the presence of non-functional p53 in resistant cells might be involved in the relative failure of cisplatin-induced apoptosis in these cells.

We recently reported the characterization of an antigen designated VRA09, identified by a monoclonal antibody and overexpressed on the surface of vincristine-resistant human ovarian carcinoma cells. In the present study, we analyze the distribution of this antigen in normal and tumor tissues. Its pattern of expression appears to differ from that described for other drug-resistance- and/or tumor-associated antigens. In normal tissues, the antigen has a restricted histological distribution and appears to be localized in mesoderm-derived tissues. In tumor tissues, VRA09 expression was mainly detected in serous ovarian tumors. Indeed, VRA09 is strongly expressed in papillary serous cystadenocarcinomas and their metastases, and more specifically in the basement membranes of serous tumors of borderline malignancy. In contrast, no immunostaining was observed in normal ovarian tissue or benign tumors. The detection of this antigen may help to identify serous ovarian tumors by distinguishing tumors of low malignancy from cystadenocarcinomas.

The prognostic value of the MDR1 gene expression in neuroblastoma (NB) was assessed in a multivariate analysis performed in a series of 84 patients (pts) taking into account the main known clinical and biological factors of the disease, i.e., age, stage, MYCN genomic content and DNA ploidy index. Twenty seven children were < 1 year (yr), 13 presented with stage I and II, 7 with stage IV-S, 17 with stage III and 47 (56%) with stage IV. Tumor specimens were obtained from involved bone marrow (n = 12) or surgical primary tumor specimens (n = 72). MDR1 gene expression was measured by Northern hybridization technique and expressed in arbitrary units (a. u.) (Goldstein et al., 1989). Analysis of MYCN genomic content and DNA ploidy index were performed by Southern blot hybridization technique and flow cytometry, respectively. Out of 84 tumor specimens 19 (23%) showed MYCN amplification (> 3 copies/haploid genome). In 24 cases (29%) no detectable MDR1 gene transcript was found (0 a.u.) whereas 42 (50%) had a value in the range 1-30 a.u., and 18 (21%) a value beyond 30 a.u.. High transcript levels were found in localized as well as in metastatic NB (NS). No significant correlation between MDR1 gene expression, age, stage, or MYCN genomic content was found In univariate analysis stage IV, age > 1 yr, MYCN amplification, diploid DNA content and high MDR1 gene transcript levels were significantly related to an increased risk of death. In multivariate analysis only stage IV, MYCN amplification and MDR1 overexpression remained significantly associated with an increased risk of death.(ABSTRACT TRUNCATED AT 250 WORDS)

To determine whether over-expression of the MDR1 gene may result from activation of its promoter by differentiating agents, the activity of the human MDR1 proximal promoter (MDR1 pp) transfected to 2 neuroblastoma lines treated with retinoic acid and forskolin was first measured using transient expression assays while the MDR1 mRNA levels were measured by Northern blots. The results indicate that retinoic acid and forskolin were able to activate the human MDR1 pp in a dose dependent manner after transfection of the MDR1pp- CAT constructs in the 2 cell models tested, i.e., SK-N-SH and IGR-N-91, a new human neuroblastoma cell line. A significant increase in MDR1 gene transcript levels was observed upon treatment with differentiation inducers in SK-N-SH but not in IGR-N-91 neuroblasts. These results suggest that the induction of the MDR1 gene promoter is necessary but not sufficient to lead to an increase in MDR1 gene transcript levels, according to the neuroblast cell line considered.

When treated in vitro with retinoic acid, many neuroblastoma cell lines undergo neuronal differentiation and show a significant increase in human multi-drug resistance gene (MDRI) transcript levels. To determine whether over-expression of the gene may result from activation of its promoter by differentiating agents, the activity of the human MDRI proximal promoter (MDRI pp) transfected to 2 neuroblastoma lines treated with retinoic acid and forskolin was first measured using transient expression assays. The MDRI gene transcript levels of these 2 lines were then measured by Northern blots. The results indicate that retinoic acid and forskolin were able to activate the human MDRI pp in a dose-dependent manner after transfection of the MDRI pp-CAT constructs in the 2 cell models tested, i.e., SK-N-SH and IGR-N-91, a new human neuroblastoma cell line. A significant increase in MDRI gene transcript levels was observed upon treatment with differentiation inducers in SK-N-SH but not in IGR-N-91 neuroblasts. These results suggest that the induction of the MDRI gene promoter is necessary but not sufficient to lead to an increase in MDRI gene transcript levels, according to the neuroblast cell line considered.

We investigated the optimal culture conditions (i.e., activation procedure, medium composition and type of culture vessel) for rapid in vitro expansion of large numbers (greater than 5 x 10(9) of blood T lymphocytes. These expanded lymphocytes can be targeted to be cytotoxic to ovarian carcinoma cells with a bispecific monoclonal antibody (BsAb) specific for CD3 and for the ovarian carcinoma-associated antigen MOv18. Both phytohemagglutinin (PHA) and monoclonal antibody (MAb) CD3 induced rapid T-cell proliferation, although the growth kinetics after PHA activation were slightly faster. A 50-fold increase in cell number was obtained after 14 and 16 days for PHA and CD3 MAb, respectively. The induction of BsAb-directed cytolysis was faster after CD3 MAb than after PHA activation of lymphocytes, but became similar around day 20. A mixture of media consisting of 78% RPMI 1640, 20% AIM-V and 2% human plasma (Mix-med) yielded better results than 100% AIM-V medium. Culture of lymphocytes in polyolefin bags, compared with tissue culture flasks, or cryopreservation did not affect lymphocyte yield and function. In most cultures the proportion of CD8+ lymphocytes increased, suggesting a growth advantage of CD8+ over CD4+ lymphocytes in this culture system. A protocol employing PHA activation, Mix-med and polyolefin bags has been used successfully to activate and expand blood lymphocytes for the first 5 patients entered into a phase-I/II clinical trial for the intraperitoneal treatment of ovarian carcinoma using CD3 x anti-MOv18 BsAb-directed T lymphocytes.

Human ovarian adenocarcinoma cells IGROV1 were induced to become resistant in vitro to Vincristine (VCR) by continuous and discontinuous stepwise exposure to the drug. Both sublines exhibited similar indexes of resistance (approx. 800). The acquisition of VCR resistance was associated with changes of morphology and with cross-resistance to Adriamycin, VP16 and actinomycin D. Addition of verapamil enhanced sensitivity of resistant cells to VCR. This multi-drug-resistant (MDR) phenotype was associated with high levels of human MDR1 gene transcripts (25- to 100-fold). In contrast to other reports, no significant amplification of the locus could be observed. Reactivity with MRK16, a monoclonal antibody (MAb) directed against the MDR 170 kDa protein, was found to be related to the level of MDR1 transcription. In addition, both OV1/VCR sublines exhibited a marked loss of tumorigenicity. Besides the chromosomal markers of OV1/p, the two resistant sublines revealed a common cytogenetic rearrangement: a deletion of the short arm of one chromosome 11: del(11)(p12). We suggest that the acquisition of this marker might be associated with the non-tumorigenic phenotype of OV1/VCR-resistant sublines.