PARP1 is activated at telomeres upon G4 stabilization: possible target for telomere-based therapy.

New anti-telomere strategies represent important goals for the development of selective cancer therapies. In this study, we reported that uncapped telomeres, resulting from pharmacological stabilization of quadruplex DNA by RHPS4 (3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate), trigger specific recruitment and activation of poly-adenosine diphosphate (ADP) ribose polymerase I (PARP1) at the telomeres, forming several ADP-ribose polymers that co-localize with the telomeric repeat binding factor 1 protein and are inhibited by selective PARP(s) inhibitors or PARP1-specific small interfering RNAs. The knockdown of PARP1 prevents repairing of RHPS4-induced telomere DNA breaks, leading to increases in chromosome abnormalities and eventually to the inhibition of tumor cell growth both in vitro and in xenografts. More interestingly, the integration of a TOPO1 inhibitor on the combination treatment proved to have a high therapeutic efficacy ensuing a complete regression of the tumor as well as a significant increase in overall survival and cure of mice even when treatments started at a very late stage of tumor growth. Overall, this work reveals the unexplored link between the PARP1 and G-quadruplex ligands and demonstrates the excellent efficacy of a multi-component strategy based on the use of PARP inhibitors in telomere-based therapy.

In vitro and in vivo antitumor efficacy of docetaxel and sorafenib combination in human pancreatic cancer cells.

The response of pancreatic cancer to treatments remains unsatisfactory, highlighting the need for more effective therapeutic regimens. Sorafenib, an orally available multikinase inhibitor, is active against different tumors, including pancreatic cancer. We studied the antitumor efficacy of sorafenib in combination with different antitumor drugs currently used in clinical practice in in vitro and in vivo experimental models of human pancreatic cancer. The cytotoxic effect of sorafenib and conventional antitumor drug combinations was evaluated in vitro in human pancreatic cancer cell lines and the efficacy of the most active combination was tested on tumor-bearing mice. Flow cytometric, Western blot and immunohistochemistry analyses were performed to investigate the mechanisms involved in the activity of single drugs and in their interaction when used in combination. Sorafenib showed a strong sequence-dependent synergistic interaction in vitro with docetaxel, which was highly dependent on the drug sequence employed. In vivo, human pancreatic cancer-xenografted mice treated with docetaxel followed by sorafenib reduced and delayed tumor growth, with complete tumor regression observed in half of the mice. This marked antitumor effect resulted in an overall increase in mouse survival of about 70% and in a complete cure in 3 of the 8 treated mice. The strong activity was also accompanied by marked apoptosis induction, inhibition of tumor angiogenesis and downregulation of ERK signalling. Our results show that the docetaxel and sorafenib combination exerts high therapeutic efficacy in experimental models of human pancreatic cancer, indicating a promising antitumor strategy for clinical use.

Targeting different signaling pathways with antisense oligonucleotides combination for cancer therapy.

The evidence that cancer development is a complex and multistep process, characterized by alterations of genes involved in the regulation of proliferation, apoptosis and angiogenesis, has led to development of new therapeutic strategies based on the use of agents able to selectively inhibit key molecules of these pathways. In particular, antisense oligonucleotides (ASOs) have proved their efficacy as targeted therapy in preclinical studies, have been well tolerated and able to modulate target protein expression in clinical studies. Although these agents have shown considerable promise for antitumoral therapy, treatment with ASOs used as single agent does not seem particularly promising because of the multigenic alterations of tumors. Based on these considerations, approaches based on the combination of ASOs targeting oncogenes involved in different molecular pathways have been investigated. Moreover, the role of this novel strategy when integrated with conventional drugs or signaling inhibitors, has been assessed. This review addresses some advances in the ASOs combination and reports the potential application of this strategy for the treatment of human cancer.

Involvement of RB gene family in tumor angiogenesis.

Angiogenesis, the development of new blood vessels from pre-existing vessels, represents a fundamental step in tumor progression and metastatization. The induction of vasculature is required for growth of the tumor mass, to ensure an adequate supply of oxygen and metabolites to the tumor beyond a critical size. Tumor angiogenesis is a highly regulated process that is controlled physiologically by the tumor microenvironment and genetically by alteration of several oncogenes or tumor suppressor genes. We will focus on recent demonstrations regarding the involvement of the retinoblastoma family proteins (phosphorylated retinoblastoma (pRb), p107 and pRb2/p130) at different levels of the angiogenic process. pRb and its homologs can regulate the expression of pro- and antiangiogenic factors, such as the vascular endothelial growth factor, through an E2F-dependent mechanism. Moreover, pRb is able to modulate also the transcriptional activity of several angiogenesis-related factors like HIF-1, Id2 and Oct-1. pRb2/p130 is required for both differentiation and mobilization of bone marrow-derived endothelial cell precursors and endothelial sprouting from neighboring vessels. The involvement of the pRb pathway in the angiogenesis process has also been demonstrated by different cellular models expressing viral oncoproteins, like human papilloma virus. Moreover, some natural and synthetic compounds demonstrate their antiangiogenetic activity with a mechanism of action involving pRb. Finally, the possible prognostic value of immunohistochemical evaluation of pRb and/or pRb2/p130 expression can represent a useful tool for the characterization of the angiogenic phenotype of specific tumor histotypes.

Involvement of hTERT in apoptosis induced by interference with Bcl-2 expression and function.

Here, we investigated the role of telomerase on Bcl-2-dependent apoptosis. To this end, the 4625 Bcl-2/Bcl-xL bispecific antisense oligonucleotide and the HA14-1 Bcl-2 inhibitor were used. We found that apoptosis induced by 4625 oligonucleotide was associated with decreased Bcl-2 protein expression and telomerase activity, while HA14-1 triggered apoptosis without affecting both Bcl-2 and telomerase levels. Interestingly, HA14-1 treatment resulted in a profound change from predominantly nuclear to a predominantly cytoplasmic localization of hTERT. Downregulation of endogenous hTERT protein by RNA interference markedly increased apoptosis induced by both 4625 and HA14-1, while overexpression of wild-type hTERT blocked Bcl-2-dependent apoptosis in a p53-independent manner. Catalytically and biologically inactive hTERT mutants showed a similar behavior as the wild-type form, indicating that hTERT inhibited the 4625 and HA14-1-induced apoptosis regardless of telomerase activity and its ability to lengthening telomeres. Finally, hTERT overexpression abrogated 4625 and HA14-1-induced mitochondrial dysfunction and nuclear translocation of hTERT. In conclusion, our results demonstrate that hTERT is involved in mitochondrial apoptosis induced by targeted inhibition of Bcl-2.

Telomerase activity, apoptosis and cell cycle progression in ataxia telangiectasia lymphocytes expressing TCL1.

Individuals affected by ataxia telangiectasia (AT) have a marked susceptibility to cancer. Ataxia telangiectasia cells, in addition to defects in cell cycle checkpoints, show dysfunction of apoptosis and of telomeres, which are both thought to have a role in the progression of malignancy. In 1-5% of patients with AT, clonal expansion of T lymphocytes carrying t(14;14) chromosomal translocation, deregulating TCL1 gene(s), has been described. While it is known that these cells can progress with time to a frank leukaemia, the molecular pathway leading to tumorigenesis has not yet been fully investigated. In this study, we compared AT clonal cells, representing 88% of the entire T lymphocytes (AT94-1) and expressing TCL1 oncogene (ATM(-) TCL1(+)), cell cycle progression to T lymphocytes of AT patients without TCL1 expression (ATM(-) TCL1(-)) by analysing their spontaneous apoptosis rate, spontaneous telomerase activity and telomere instability. We show that in ATM(-) TCL1(+) lymphocytes, apoptosis rate and cell cycle progression are restored back to a rate comparable with that observed in normal lymphocytes while telomere dysfunction is maintained.

Bcl-2 has differing effects on the sensitivity of breast cancer cells depending on the antineoplastic drug used.

The aim of this paper was to evaluate the role of bcl-2 in the susceptibility of the MCF7 ADR human breast carcinoma line overexpressing the P-170 glycoprotein (P-170) to various drugs. The sensitivity to four multidrug resistance (MDR)-related drugs (doxorubicin (ADR), vincristine (VCR), vinblastine (VBL), actinomycin D (ACTD)) and three MDR-non-related drugs (cisplatin (DDP), bischloroethylnitrosourea (BCNU), 5-fluorouracil (5-FU)) was evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay in three bcl-2-overexpressing clones obtained from the MCF7 ADR line. We found that the bcl-2-overexpressing clones show increased resistance to DDP and BCNU, while no difference to 5-FU were observed between the control cells and bcl-2 transfectants. Surprisingly, bcl-2-overexpressing clones displayed an increased sensitivity compared with the control cells to the MDR-related drugs ADR, VCR, VBL and ACTD. Focusing on DDP and ADR, we found that the increased resistance of the bcl-2 transfectants to DDP was correlated to their ability to prevent apoptosis, while the enhanced sensitivity to ADR was associated with an increased ADR accumulation and a decreased ADR efflux. Moreover, while bcl-2 overexpression does not induce changes in P-170 glycoprotein expression, it did induce a reduction of the adenosine triphosphate (ATP) levels and basal protein kinase C (PKC) activity, both of which have a crucial role in the regulation of the MDR phenotype. In conclusion, the effect of bcl-2 on antineoplastic sensitivity observed in this study underscores the idea that bcl-2 may have distinct biological effects depending on the anticancer drug used.

relA over-expression reduces tumorigenicity and activates apoptosis in human cancer cells.

We previously demonstrated that bcl-2 over-expression increases the malignant behaviour of the MCF7 ADR human breast cancer cell line and enhances nuclear factor-kappa B (NF-kappa B) transcriptional activity. Here, we investigated the direct effect of increased NF-kB activity on the tumorigenicity of MCF7 ADR cells by over-expressing the NF-kappa B subunit relA/p65. Surprisingly, our results demonstrated that over-expression of relA determines a considerable reduction of the tumorigenic ability in nude mice as indicated by the tumour take and the median time of tumour appearance. In vitro studies also evidenced a reduced cell proliferation and the activation of the apoptotic programme after relA over-expression. Apoptosis was associated with the production of reactive oxygen species, and the cleavage of the specific substrate Poly-ADP-ribose-polymerase. Our data indicate that there is no general role for NF-kappa B in the regulation of apoptosis and tumorigenicity. In fact, even though inhibiting NF-kappa B activity has been reported to be lethal to tumour cells, our findings clearly suggest that an over-induction of nuclear NF-kappa B activity may produce the same effect.

A role for c-myc in DNA damage-induced apoptosis in a human TP53-mutant small-cell lung cancer cell line.

Based on the role of p53 in the control of apoptosis following DNA damage, the status of the TP53 gene has been implicated as a major determinant of tumour responsiveness to cytotoxic therapies. In spite of the high frequency of TP53 mutations, small-cell lung cancer (SCLC) is recognised as one of the most chemoresponsive solid tumours. Since the relevance of the TP53 gene status in the modulation of tumour responsiveness is dependent on the molecular/biological context, in the present study, we have examined the relationship between chemosensitivity and susceptibility to apoptosis of a TP53-mutant human SCLC cell line. The cell line, in spite of TP53 mutation, retained an efficient response to genotoxic stress as documented by cells ability to modulate the p53 protein, arrest in the G1 and G2 phases of the cell cycle and its marked susceptibility to apoptosis following treatment with DNA damaging agents. Exposure to DNA-damaging agents caused an increase of c-Myc, a DNA damage-responsive transcription factor. An analysis of damage-induced apoptosis in the presence of an anti-Fas/CD95 inhibitory antibody indicated that Fas/CD95 was not required for the apoptotic response. The results support an implication of c-myc in sensitising cells to apoptosis, since inhibition of c-Myc expression with an antisense oligodeoxynucleotide (AS-ODN) almost abolished the drug-induced apoptotic response. In conclusion, the present results support a role for c-myc in the induction of apoptosis by genotoxic stress in the absence of a functional p53 and provide new insights into the mechanisms that may influence apoptosis in TP53-mutant cells. Elucidation of this pathway and of the possible cooperation with p53-dependent mechanisms may provide a basis for therapeutic intervention.

Bcl-2 overexpression decreases BCNU sensitivity of a human glioblastoma line through enhancement of catalase activity.

The aim of this study was to evaluate the role of bcl-2 in 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) sensitivity of the ADFS human glioblastoma cell line in vitro and in vivo. To this end, the ADFS line expressing a low level of the bcl-2 protein was transfected with a bcl-2 expression vector. We found that bcl-2 overexpressing clones were less sensitive to in vitro BCNU treatment than the control clone. Cell cycle analysis demonstrated that while BCNU induced a consistent block in S/G2-M phases of the cell cycle in the control clone, it did not affect the cell cycle phase distribution of the two bcl-2 transfectants. The different sensitivity to BCNU was unrelated to the ability of bcl-2 to inhibit apoptosis, while bcl-2 appeared to protect bcl-2 transfectants from BCNU toxicity through an increase of catalase activity. The ability of the catalase inhibitor, sodium azide, to increase the BCNU sensitivity of the bcl-2 transfectants to levels of the BCNU-treated control clone substantiated the role of the catalase activity. The effect of bcl-2 in reducing sensitivity to BCNU was also confirmed by in vivo experiments. Xenografts of bcl-2 overexpressing tumors were less sensitive to BCNU treatment than xenografts originating from control cells.

p53 nuclear accumulation and multiploidy are adverse prognostic factors in surgically resected stage II colorectal cancers independent of fluorouracil-based adjuvant therapy.

To identify the prognostically highest risk patients, DNA content and p53 nuclear or cytoplasmic accumulation, evaluated by monoclonal antibody DO7 and polyclonal antibody CM1, were determined in 94 surgically resected stage II (Dukes B2) colorectal cancers, treated or not with adjuvant 5-fluorouracil-based chemotherapy. Sixty-one (65%) of the tumors were aneuploid, 16 (17%) of which had a multiploid DNA content; 50 (53%) displayed DO7 nuclear p53 accumulation, and 44 (47%) showed cytoplasmic CM1 positivity. In multivariate analysis, only multiploidy and p53 nuclear positivity emerged as independent prognostic indicators of a poorer outcome. Positivity for p53 was associated with shorter survival in 5-fluorouracil-treated and untreated patients. Therefore, in patients with Dukes B2 colorectal cancer, a biologic profile based on the combined evaluation of DNA multiploidy and p53 status can provide valuable prognostic information, identifying patients to be enrolled in alternative, more aggressive therapeutic trials.

Encapsulation of c-myc antisense oligodeoxynucleotides in lipid particles improves antitumoral efficacy in vivo in a human melanoma line.

Phosphorothioate c-myc antisense oligodeoxynucleotides [S]ODNs (free INX-6295) were encapsulated in a new liposome formulation and the antitumor activity was compared to the unencapsulated antisense in a human melanoma xenograft. The systemic administration of INX-6295 encapsulated in stabilized antisense lipid particles (SALP INX-6295) improved plasma AUC (area under the plasma concentration-time curve) and initial half-life of free INX-6295, resulting in a significant enhancement in tumor accumulation and improvement in tumor distribution of antisense oligodeoxynucleotides. Animals treated with SALP INX-6295 exhibited a prolonged reduction of c-myc expression, reduced tumor growth and increased mice survival. When administered in combination with cisplatin (DDP), SALP INX-6295 produced a complete tumor regression in approximately 30% of treated mice, which persisted for at least 60 days following the first cycle of treatment. Finally, the median survival of mice treated with DDP/SALP INX-6295 increased by 105% compared to 84% for animals treated with the combination DDP/free INX-6295. These data indicate that the biological activity and the therapeutic efficacy of c-myc antisense therapy may be improved when these agents are administered in lipid-based delivery systems.

c-Myc down-regulation increases susceptibility to cisplatin through reactive oxygen species-mediated apoptosis in M14 human melanoma cells.

Our aim in this work was to define the role of c-Myc in the susceptibility to cisplatin [cis-diamminedichloroplatinum(II) (CDDP)] in human melanoma cells. Two M14 melanoma cell clones obtained by transfection and expressing six to ten times lower c-Myc protein levels than the parental cells and the control clone were employed. Analysis of survival curves demonstrates an increase in CDDP sensitivity in c-Myc low-expressing clones if compared with the control clone and the parental line. The enhanced sensitivity is unrelated to the impairment in enzymatic DNA repair activity. Cell cycle analysis demonstrates that although the control clone is able to completely recover from the CDDP-induced S-G(2)/M block, this arrest is prolonged in c-Myc low-expressing clones and a fraction of cells undergoes apoptosis. Although no changes in P53, Bax, Bcl-2, and Bcl-x(L/S) protein levels are observed, apoptosis is associated with the formation of reactive oxygen species (ROS), activation of caspase-1, caspase-3 and cleavage of the specific caspase substrate poly-ADP-ribose polymerase. The use of the antioxidant N-acetyl cysteine and caspase inhibitors prevents CDDP-induced apoptosis in c-Myc low-expressing clones, demonstrating that ROS, caspase-1, and caspase-3 are required for apoptotic cell death. Moreover, ROS generation depends on caspase-1-like activation because the Ac-YVAD-cho inhibitor abrogates CDDP-induced ROS in the c-Myc low-expressing clones.

c-Myb and Bcl-x overexpression predicts poor prognosis in colorectal cancer: clinical and experimental findings.

The aim of this study was twofold: to assess the relationship between c-Myb and Bcl-x expression and to evaluate the prognostic significance of their expression in colorectal carcinoma (CRC) patients. Analysis of tumors from 91 CRC patients for expression of c-Myb and Bcl-x revealed a significant relationship between these two proteins. Kaplan-Meier's analysis showed an increased risk of relapse and death in patients whose tumor specimens displayed high c-Myb levels and Bcl-x positivity. Similar results were also observed excluding Dukes' D patients. Molecular analysis using three c-Myb-overexpressing LoVo clones indicated that c-Myb overexpression was accompanied by up-regulation of Bcl-x(L) protein and mRNA. Tumors originating from these clones injected in nude mice were significantly larger than those formed in mice injected with parental or vector-transfected LoVo cells. Moreover, tumors derived from parental and control vector-transfected but not from c-Myb-overexpressing LoVo cells showed high frequency of apoptotic cells. These results provide direct evidence of an association between c-Myb and Bcl-x expression and suggest that expression of both molecules might be a useful prognostic marker in CRC.

Bcl-2 overexpression and hypoxia synergistically act to modulate vascular endothelial growth factor expression and in vivo angiogenesis in a breast carcinoma line.

We have previously demonstrated that bcl-2 overexpression enhances the metastatic potential of the MCF7 ADR human breast cancer cell line resistant to adriamycin by inducing metastasis-associated properties. To further elucidate the relationship between bcl-2 expression and the metastatic potential of the MCF7 ADR line, we evaluated whether bcl-2 could be also involved in the modulation of the angiogenic phenotype. Four bcl-2-overexpressing clones, a control transfectant clone, and the MCF7 ADR parental line were used for in vitro and in vivo experiments. Bcl-2 overexpression enhanced the synthesis of the hypoxia-stimulated VEGF protein and mRNA. Northern blot analysis demonstrated an increased VEGF mRNA expression in bcl-2-overexpressing clones, and reverse transcription-polymerase chain reaction showed higher levels of the VEGF(121) and VEGF(165) mRNA isoforms, which are the most active in eliciting angiogenesis. When incorporated into matrigel, supernatants of bcl-2-transfected cells cultured under hypoxic conditions induced an increased angiogenic response in C57BL/6 mice compared with that of control clone. Tumors from bcl-2 transfectants demonstrated increased VEGF expression and neovascularization as compared to the parental line, whereas the apoptosis in in vivo xenografts was similar in control and bcl-2 transfectants. The effect of bcl-2 on angiogenesis was not mediated by p53 protein. These results demonstrate that bcl-2 and hypoxia can act synergistically to modulate VEGF expression and the in vivo angiogenic response in the MCF7 ADR line.

Increased TGFbeta type II receptor expression suppresses the malignant phenotype and induces differentiation of human neuroblastoma cells.

TGFbeta can modulate neuroblastoma (NB) cell proliferation and differentiation in vitro. In this study we used a NB cell line (LAN-5) which has been shown to partially respond to TGFbeta and to present high levels of TGFbeta receptor type I and low levels of receptor type II (TbetaRII) on the cell surface. To evaluate the role of TbetaRII in mediating TGFbeta effects, LAN-5 cells were transfected with an expression vector containing the human full-length TbetaRII cDNA or with the empty vector pcDNA3. Compared to control CLV3 cells (transfected with empty plasmid) and parental LAN-5 cells, isolated neomycin-resistant clones (CL1 and CL3) expressed higher levels of TbetaRII, had reduced cell growth rate in vitro, and were unable to form tumors in vivo. Furthermore, isolated clones modified their morphology, assuming a terminally differentiated neuronal phenotype. Immunocytochemical staining demonstrated a basal increased expression of neural-specific markers, such as axonal growth-associated protein (GAP43) and neurofilaments (NF200). TGFbeta treatment further increased the synthesis of NF200 and GAP43 in the transfected clones as revealed by Western blot analysis. These data indicate that TbetaRII overexpression potentiates the TGFbeta signal transduction pathway, reverting NB cell neoplastic phenotype with the reduction of proliferation rate and the induction of terminal maturation.

Enhanced anti-tumor effects with microencapsulated c-myc antisense oligonucleotide.

A phosphorothioate c-myc antisense oligonucleotide was complexed with zinc and encapsulated into injectable biodegradable microspheres. The efficacy of this novel formulation was compared with intravenous administration of the unencapsulated drug in human melanoma and leukemia xenografts in immunocompromised mice. The microencapsulated formulation was more effective as shown by reduced tumor growth, a decreased number of metastases, reduced c-myc expression, and increased survival in the melanoma model, and decreased metastatic potential and increased survival in the leukemia model. These results show that, as has been demonstrated previously with protein and peptide drugs, greater therapeutic efficacy can be obtained when antisense oligonucleotides are delivered from sustained-release formulations.

Evaluation of multiple bio-pathological factors in colorectal adenocarcinomas: independent prognostic role of p53 and bcl-2.

About 40% of patients with colorectal carcinoma will develop local or distant tumour recurrences. Integrated analyses of bio-pathological markers, predictive of tumour aggressiveness, may offer a more rational approach to planning adjuvant therapy. To this end, we analysed the correlation between p53 accumulation, Bcl-2 expression, DNA ploidy, cell proliferation and conventional clinico-pathological parameters by testing the prognostic significance of these variables in a series of 171 colorectal carcinoma patients with long-term follow-up. The relationships among the various bio-pathological parameters, analysed by multiple correspondence analysis, showed 2 different clinico-biological profiles. The first, characterised by p53 negativity, Bcl-2 positivity, diploidy, low percentage of cells in S-phase (%S-phase), a low Ki-67 score, is associated with Dukes' A-B stage, well differentiated tumours and lack of relapse. The second, defined by p53 positivity, Bcl-2 negativity, aneuploidy, high %S-phase and elevated Ki-67 score, correlates with Dukes' C-D stage, poorly differentiated tumours and presence of relapse. When these parameters were examined according to Kaplan-Meier's method, significantly shorter disease-free (DFS) and overall survival (OS) were also observed in patients bearing p53 positive and Bcl-2 negative tumours, in Dukes' B stage. In multivariate analysis, p53 accumulation and Bcl-2 expression emerged as independent predictors of a worse and better clinical outcome, respectively. Our results indicate that, in colorectal adenocarcinomas, a biological profile, based on the combined evaluation of p53 and Bcl-2, may be useful for identifying high risk patients to be enrolled in an adjuvant setting, mainly in an early stage of the disease. Int. J. Cancer (Pred. Oncol.) 84:545-552, 1999.

Increase of cisplatin sensitivity by c-myc antisense oligodeoxynucleotides in a human metastatic melanoma inherently resistant to cisplatin.

In this study, we evaluated the role of the c-myc oncogene in response to cisplatin (DDP) treatment using two melanoma lines derived from the primary tumor (LP) and metastatic lymph node (LM) of the same patient. These cell lines, which retain the phenotypic profile of the original tumors, were studied for growth behavior, expression of c-Myc oncoprotein, and HLA-I antigen. The LM line shows a higher tumorigenic ability, an increased expression of c-Myc protein, and a lack of HLA-I antigen, compared with the LP line. In addition, LP tumor was relatively sensitive to DDP administration, whereas LM tumor was resistant to DDP treatment. To verify whether the increased c-Myc expression observed in the LM line might be responsible for DDP resistance, a c-myc antisense phosphorothioate oligodeoxynucleotide ([S]ODN) was used to down-regulate c-Myc expression. The administration of DDP plus c-myc antisense [S]ODNs produced a decrease in c-Myc protein levels of approximately 50%, accompanied by a tumor weight inhibition of 65%, similar to that obtained when the sensitive line was treated with DDP alone (tumor weight inhibition = 70%). Analysis of apoptosis demonstrated that the sensitivity to DDP of the LP line was related to the ability of tumor cells to undergo apoptosis. Conversely, DDP treatment was not able to induce apoptosis in the LM line, whereas apoptosis was evident both after treatment with c-myc antisense [S]ODNs alone and, more extensively, in combination with DDP. Taken together, these results clearly indicate an important role of c-myc oncogene in the resistance of melanoma to DDP and demonstrate that treatment with c-myc antisense [S]ODN sensitizes a human melanoma line to DDP treatment.

Increase of BCNU sensitivity by wt-p53 gene therapy in glioblastoma lines depends on the administration schedule.

In this article, we investigated the effect induced by the reintroduction of wild-type p53 (wt-p53) protein on BCNU sensitivity in the ADF glioblastoma line. Using a wt-p53 recombinant adenovirus (Ad-p53), we demonstrated that exogenous wt-p53 expression was able to increase the sensitivity to BCNU in ADF cells. Interestingly, this effect was more evident when Ad-p53 infection was performed after BCNU treatment compared with the opposite sequence. To understand the biological basis of these different behaviors, we analyzed the cell cycle of the differently treated cells. We found that Ad-p53 infection induced a persistent accumulation of cells in the G0/G1 phase while, as expected, BCNU induced a block in the G2-M phase. Ad-p53-->BCNU sequence did not significantly modify the cell cycle profile in respect of Ad-p53 infected cells. In contrast, BCNU-->Ad-p53 sequence provoked G2-M arrest similar to that observed after treatment with BCNU alone, but prevented the later recovery of the cells through the cell cycle, by driving the cells to apoptotic death. These results demonstrate that the administration sequence is important to increase drug sensitivity. To generalize the phenomenon observed on ADF line, the antiproliferative effect of the two different schedules was analyzed on other glioblastoma lines (A172, CRS-A2, U373MG) with different BCNU sensitivity and p53 status. The data obtained confirm that the wt-p53 gene transfer enhances BCNU sensitivity in glioblastoma cells depending on the administration sequence.