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Photodynamic therapy (PDT) is a treatment for cancer based on the photosensitization of tumor cells by photosensitive drugs and their subsequent destruction on exposure to light of particular wavelength. The combination of drug uptake in malignant tissues and selective delivery of laser-generated light provides an effective therapy with efficient tumor citotoxicity and minimal normal tissue damage. Since immune response of the host is important in the control of tumor growth and spreading, PDT is able to increase the antitumor immunity. In our laboratory we examined the antitumor effect of combination of PDT, with photoactivated M-THPC (meta-tetrahydroxyphenylchlorin, FOSCAN, Temoporphirin), adoptive immunotherapy, with immune lymphocytes, and chemotherapy on advanced murine tumors. Mice bearing L1210 tumor were treated at day +4 with Navelbine (NVB 1mg/Kg), at day +5,+6 with PDT (0.3mg/Kg of mTHPC and 100mW/cm(2) x 200'' of exposure of laser light), and at day + 7 with immune lymphocytes(IL), collected from mice pretreated with PDT(2x10(7) cells). The results show that the combination NVB + PDT + IL demonstrates a significant synergistic antitumor effect while the chemotherapy treatment with low dose of the drug is uneffective. The same positive results were obtained with the combination of Cisplatin (CDDP 0.5mg/Kg), PDT and IL, while the CDDP treatment alone is completely uneffective. In conclusion, these results suggest that it is possible to completely cure animals bearing advanced tumors, with a combined therapy, PDT + adoptive immunotherapy + low dose chemotherapy.

In the 3′-untranslated region, the destabilizing adenine-uridine (AU)-rich elements (AREs) control the expression of several transcripts through interactions with ARE-binding proteins (AUBPs) and RNA degradation machinery. Although the fundamental role for AUBPs and associated factors in eliciting ARE-dependent degradation of cognate mRNAs has been recently highlighted, the molecular mechanisms underlying the specific regulation of individual mRNA turnover have not yet been fully elucidated. Here we focused on the post-transcriptional regulation of bcl-2 mRNA in human cell lines under different conditions and genetic backgrounds. In the context of an AUBPs silencing approach, HuR knockdown reduced the expression of endogenous bcl-2, whereas unexpectedly, a bcl-2 ARE-reporter transcript increased significantly, suggesting that HuR expression has opposite effects on endogenous and ectopic bcl-2 ARE. Moreover, evidence was provided for the essential, specific and dose-dependent role of the Bcl-2 protein in regulating the decay kinetics of its own mRNA, as ascertained by a luciferase reporter system. Altogether, the data support a model whereby the Bcl-2 protein is the major determinant of its own ARE-dependent transcript half-life in living cells and its effect overcomes the activity of ARE-binding proteins.

Modulation of mRNA stability by regulatory cis-acting AU-rich elements (AREs) and ARE-binding proteins is an important posttranscriptional mechanism of gene expression control. We previously demonstrated that the 3'-untranslated region of BCL-2 mRNA contains an ARE that accounts for rapid BCL-2 down-regulation in response to apoptotic stimuli. We also demonstrated that the BCL-2 ARE core interacts with a number of ARE-binding proteins, one of which is AU-rich factor 1/heterogeneous nuclear ribonucleoprotein D, known for its interaction with mRNA elements of others genes. In an attempt to search for other BCL-2 mRNA-binding proteins, we used the yeast RNA three-hybrid system assay and identified a novel human protein that interacts with BCL-2 ARE. We refer to it as TINO. The predicted protein sequence of TINO reveals two amino-terminal heterogeneous nuclear ribonucleoprotein K homology motifs for nucleic acid binding and a carboxyl-terminal RING domain, endowed with a putative E3 ubiquitin-protein ligase activity. In addition the novel protein is evolutionarily conserved; the two following orthologous proteins have been identified with protein-protein BLAST: posterior end mark-3 (PEM-3) of Ciona savignyi and muscle excess protein-3 (MEX-3) of Caenorhabditis elegans. Upon binding, TINO destabilizes a chimeric reporter construct containing the BCL-2 ARE sequence, revealing a negative regulatory action on BCL-2 gene expression at the posttranscriptional level.

The serine/threonine kinase mTOR, the major sensor of cell growth along the PI3K/Akt pathway, can be activated by agents acting on microtubules. Damaged microtubules induce phosphorylation of the Bcl-2 protein and lower the threshold of programmed cell death, both of which are inhibited by rapamycin. In HEK293 cells expressing Akt mutants, the level of Bcl-2 phosphorylation and the threshold of apoptosis induced by taxol or by nocodazole are significantly modified. In cells expressing dominant-negative Akt (DN-Akt), Bcl-2 phosphorylation and p70S6KThr421/Ser424 phosphorylation induced by taxol or nocodazole were significantly enhanced as compared to cells expressing constitutively active Akt (CA-Akt) and inhibited by rapamycin. Moreover, DN-Akt cells were more sensitive to antitubule agents than CA-Akt cells. In nocodazole-treated HEK293 cells sorted according to cell cycle, the p70S6KThr421/Ser424 phosphorylation was associated to the G2/M fraction. More relevant, nocodazole inhibited, in a dose-response manner, mTOR phosphorylation at Ser2448. This activity, potentiated in DN-Akt cells, was not detectable in CA-Akt cells. Our results suggest that death signals originating from damaged microtubules in G2/M can compete with G1 survival pathways at the level of mTOR. These findings have implications for cancer therapy and drug resistance.

Photodynamic therapy (PDT) is based on the administration of tumor-localizing photosensitizers followed by light exposure of the tumor mass. The photocytotoxic effects are mainly caused by the generation of singlet oxygen. Recently, PDT has been proposed for use in combination with anticancer chemotherapy with a view to exploiting any additive antitumor effect. We investigated the effect of PDT with photoactivated aluminum disulfonated phthalocyanine (AlS2Pc) combined with the antiblastic drugs Adriamycin (ADR) and cisplatinum (CDDP) on murine tumors. Mice bearing L1210 leukemia and P388 lymphoma were treated with ADR or CDDP and subsequently treated with PDT. Low chemotherapy doses were ineffective, but the combination of antiblastic drugs + PDT had a significantly additive antitumor effect. In conclusion, with this combined therapy we were able to greatly reduce the effective doses of antiblastic drugs, thus lowering their toxic effects on normal host tissues.

The effect of an antisense oligonucleotide to the K+ channel coding mKv1.1 mRNA on antinociception induced by the tricyclic antidepressants, clomipramine (20–35 mg kg−1 s.c.) and amitriptyline (10–25 mg kg−1 s.c.), was investigated in the mouse hot-plate test. Antisense oligonucleotide (0.5-1.0-2.0-3.0 nmol per i.c.v. injection) produced a dose-dependent inhibition of clomipramine and amitriptyline antinociception 72 h after the last i.c.v. injection. The sensitivity to both analgesic drugs returned 7 days after antisense oligonucleotide injection, indicating the absence of irreversible damage or toxicity. Treatment with a degenerated oligonucleotide did not modify the clomipramine- and amitriptyline-induced antinociception in comparison with that in naive (unpretreated controls), vector and saline i.c.v.-injected mice. A quantitative reverse transcription-polymerase chain reaction (RT-PCR) study demonstrated a reduction in mRNA levels only in the antisense oligonucleotide treated group. Antisense oligonucleotide, degenerated oligonucleotide or vector pretreatment, in the range of doses used, did not produce any behavioural impairment as revealed by the mouse rotarod and hole-board tests. The present results indicate that modulation of the mKv1.1 K+ channel plays an important role in the central analgesia induced by the tricyclic antidepressants, clomipramine and amitriptyline.

To assess the prognostic value of tumor proliferative activity, 89 patients with operable non-small cell lung cancer were studied. Tumor samples were obtained during surgery and cell kinetics were analyzed by the in vitro thymidine labelling index (TLI). The overall median TLI (2.9) was used to identify two subsets of patients with high and low proliferating tumors. In univariate analysis survival was significantly longer in patients with lower TLI (P = 0.047) and with stage I-II (P = 0.003) and T1-T2 tumors (P = 0.043). In multivariate analysis, stage was the most important prognostic parameter (P = 0.004). The risk of death for patients with TLI higher than 2.9 was increased (hazard ratio = 2.01, CI = 0.96-4.27).

Thirty-nine patients with locally advanced breast cancer (T3b-4, N1-3 or inflammatory carcinoma) received 3 cycles of induction chemotherapy with estrogenic recruitment before surgery. The therapeutic regimen consisted of diethylstilbestrol (DES) orally on days 1-3, 5-Fluorouracil + Doxorubicin + Cyclophosphamide on day 4 q 21 days (DES-FAC). After surgery 6 additional cycles of chemotherapy (3 DES-FAC alternating with 3 DES-CMF with Methotrexate + F and C as in FAC) were administered. The objective response rate was 71.8% with 15.4% CR, and 56.4% PR; after surgery 36/39 (92.3%) patients were rendered disease-free. So far, 13 of 26 patients in stage IIIb have relapsed (9 of 13 with inflammatory carcinomas). Three-year survival and progression-free survival are 60% and 53.5%, respectively. Twenty-three of the 39 patients were subjected to serial tumor biopsies during the first DES-FAC regimen to allow for tumor-cell kinetic studies during DES and chemotherapy. A significant estrogenic recruitment occurred in 16 patients (69.6%), irrespective of estrogen-receptor status. At surgery, 3-4 weeks after induction chemotherapy, tumor proliferative activity was significantly depressed in comparison to basal values. These results indicate that breast cancer cells can be recruited in vivo with DES and that chemotherapy following estrogenic stimulation is effective and feasible with acceptable toxicity.

Background CDK5R1 plays a central role in neuronal migration and differentiation during central nervous system development. CDK5R1 has been implicated in neurodegenerative disorders and proposed as a candidate gene for mental retardation. The remarkable size of CDK5R1 3'-untranslated region (3'-UTR) suggests a role in post-transcriptional regulation of CDK5R1 expression. Results The bioinformatic study shows a high conservation degree in mammals and predicts several AU-Rich Elements (AREs). The insertion of CDK5R1 3'-UTR into luciferase 3'-UTR causes a decreased luciferase activity in four transfected cell lines. We identified 3'-UTR subregions which tend to reduce the reporter gene expression, sometimes in a cell line-dependent manner. In most cases the quantitative analysis of luciferase mRNA suggests that CDK5R1 3'-UTR affects mRNA stability. A region, leading to a very strong mRNA destabilization, showed a significantly low half-life, indicating an accelerated mRNA degradation. The 3' end of the transcript, containing a class I ARE, specifically displays a stabilizing effect in neuroblastoma cell lines. We also observed the interaction of the stabilizing neuronal RNA-binding proteins ELAV with the CDK5R1 transcript in SH-SY5Y cells and identified three 3'-UTR sub-regions showing affinity for ELAV proteins. Conclusion Our findings evince the presence of both destabilizing and stabilizing regulatory elements in CDK5R1 3'-UTR and support the hypothesis that CDK5R1 gene expression is post-transcriptionally controlled in neurons by ELAV-mediated mechanisms. This is the first evidence of the involvement of 3'-UTR in the modulation of CDK5R1 expression. The fine tuning of CDK5R1 expression by 3'-UTR may have a role in central nervous system development and functioning, with potential implications in neurodegenerative and cognitive disorders.