Bioavailability of antisense oligonucleotides in neuroblastoma cells: comparison of efficacy among different types of molecules.

To evaluate the real effectiveness of various chemical modifications in enhancing the ability of antisense molecules to inhibit gene expression, the toxicity, stability, uptake, and intracellular localization of an identical sequence, synthetized either with a phosphodiester or a phosphorothioate backbone, with or without a cholesteryl moiety linked to the 3'-end, were compared in three different human neuroblastoma cell lines. The toxicity, assessed by inhibition of cell viability, greatly depend on the presence of the lipid moiety and to a less extent on the cell line used. At high doses all the antisenses caused a necrotic lysis of plasma membranes. Typical features of apoptotic cell death were never observed. The presence of the lipid moiety enhanced the uptake of antisense molecules while the phosphorothioate backbone, as expected, conferred higher stability. At late times, therefore, the combination of lipid conjugation and phosphorothioate backbone seems to be the most effective in obtaining a consistent antisense accumulation inside the cells. The presence of the cholesteryl moiety also caused a stronger association of the antisense to membraneous compartments, so that a quite different biodistribution occurred among the four antisenses tested. However, the actual amount of antisense molecules found inside NB cells was low in all the conditions tested. Only following cellular permeabilization a significant uptake was obtained, making the use of delivery system mandatory to achieve an efficient inhibition of highly expressed genes.

Increase of metaiodobenzylguanidine uptake and intracellular half-life during differentiation of human neuroblastoma cells.

Iodine-labelled metaiodobenzylguanidine (MIBG) is a radiopharmaceutical used for diagnostic imaging and targeted radiotherapy of neuroendocrine tumors. We previously reported that the ability of a neuroblastoma (NB) cell line, LAN-5, to accumulate MIBG was powerfully stimulated by interferon-gamma (IFN-gamma), a well-known NB differentiation-promoting agent. To extend the above findings, we have investigated 5 NB cell lines for their ability to accumulate 125I-MIBG in basal conditions or after various combinations of differentiative stimuli. Our results show that association of IFN-gamma and tumor necrosis factor-alpha boosts MIBG uptake in the early times of incubation in LAN-5 and GI-LI-N cells, while both SK-N-SH and SK-N-BE(2)c cells are strongly stimulated by co-treatment with IFN-gamma and all-trans retinoic acid. Moreover, although only LAN-5 and GI-LI-N cells are sensitive to IFN-gamma alone, the combination of IFN-gamma and IFN-alpha causes a synergistic increase in MIBG uptake in all the NB cell lines tested. From experiments on MIBG release we conclude that no intracellular storage within specialized structures took place during differentiation. The observed enhancement in MIBG accumulation results from an increased uptake of the drug only. This conclusion was confirmed by analyzing MIBG-transporter gene expression, which was increased in cells subjected differentiative regimens. According to these findings, inducing differentiation of NB cells in vitro appears to improve their MIBG incorporation ability powerfully.

Induction of 2.5 OAS gene expression and activity is not sufficient for IFN-gamma-induced neuroblastoma cell differentiation.

We showed earlier that interferon-gamma is a powerful inducer of differentiation of human neuroblastoma (NB) cells. Although 2',5' oligo-adenylate synthetase (2,5 OAS) may play a role in mediating the anti-proliferative and/or differentiative effects of interferons (IFNs), direct evidence is lacking. We have investigated gene and protein expression of the 4 different 2,5 OAS isoforms and their cumulative enzymatic activity in a previously characterized IFN-gamma-sensitive human NB cell line, LAN-5. Analysis of total and poly(A)+ RNA by Northern blot and RT-PCR indicated that expression of the mRNA coding for the 40-, 46-and 69-kDa isoforms was induced in a time- and dose-dependent manner, reaching a maximum after a 36-hr treatment with 1000 IU/ml of IFN-gamma. In the absence of treatment, only the mRNA for the 69-kDa isoform was detectable by RT-PCR. Inhibition of transcription with actinomycin D showed that 2,5 OAS mRNA was quite stable, with a half-life of about 4 hr. With respect to the protein content, no 2,5 OAS isoform was present in proliferating LAN-5 cells; following IFN-gamma treatment, the 100-, 69-and 46-kDa isoforms became detectable. Accordingly, 2,5 OAS enzymatic activity, virtually undetectable in untreated LAN-5 cells, increased up to 132 pmol oligoadenylate/micrograms protein/hr after 48 hr of treatment, then slowly decreased, remaining detectable up to 96 hr. However, the 2,5 OAS proteins required an exogenous activation by synthetic dsRNA to exert enzymatic activity. It is therefore conceivable that they do not play a biological role in NB cell functions. Moreover, an increase in 2,5 OAS enzymatic activity was also observed in NB cells resistant to the differentiation-promoting activity of IFN-gamma, further suggesting that 2,5 OAS induction was not sufficient to trigger IFN-gamma-dependent neuronal maturation. Furthermore, other differentiation-inducing agents, such as retinoic acid and cytosine arabinoside, or complete proliferative arrest produced by serum deprivation, failed to enhance 2,5 OAS activity, thus indicating that the 2,5 OAS system is not directly involved in mediating other differentiative pathways of NB cells.

Self-limiting, cell type-dependent replication of an integrase-defective human immunodeficiency virus type 1 in human primary macrophages but not T lymphocytes.

Integration of retroviral DNA into the host cell genome, catalyzed by the integrase (IN) protein, is thought to be required for replication. We show here that one IN-minus defective mutant of human immunodeficiency virus type 1 (HIV-1) is able to replicate in macrophages but not in peripheral blood lymphocytes (PBLs). Replication of the HIV-1 defective mutant, however, was inefficient and self-limiting. The absence of integration in the HIV-1 IN mutant in contrast to the wild-type implies that the replication of the IN mutant depends on the transcription of the extrachromosomal forms of viral DNA. In both PBLs and macrophages circular forms of DNA were detected at significant levels, indicating that the lack of a complete functional IN protein does not preclude nuclear import of HIV-1 DNA. Cell-associated p24 was absent in the IN-defective-infected PBLs, suggesting a transcriptional block of the extrachromosomal forms of HIV-1. These results show the existence of different strategies for HIV-1 replication depending upon the cell type, and indicate the necessity of integration of viral DNA for the self-maintained progression of the infection.

[Neuroblastoma as a model for the in-vitro study of differentiated therapy]. / Il neuroblastoma come modello di studio in vitro della terapia differenziativa.

Differentiation of human neuroblastoma (NB) cells is a very interesting biologic event, providing useful insights in both basic neurobiology and clinical management of this malignancy. Investigation of in vitro NB differentiation exploits several NB cell lines that can be induced to differentiate by an array of natural or synthetic chemicals, as well as biological factors such as some cytokines. The hallmarks of neuronal differentiation are represented by a partial or complete block of cell proliferation, morphological alterations and acquisition of biological features typical of mature neurons (for example, induced synthesis and storage of monoamines and neuropeptides, expression of peculiar cytoskeletal proteins and membrane antigens). The possibility to transfer the differentiative approach to the treatment of NB patients opens exciting therapeutic perspectives.

Cloning and sequencing of isoform-specific regions of human Ca2+-independent protein kinase C (PKC)-encoding genes.

The cloning and sequencing of the isoform-specific regions of the human Ca2+-independent protein kinase C-encoding genes is described. These clones will serve as correct size probes for screening human genomic or cDNA libraries and isolating full-length clones.

Uncoordinate induction and differential regulation of HLA class-I and class-II expression by gamma-interferon in differentiating human neuroblastoma cells.

Recombinant gamma-interferon (IFN-gamma) has recently been shown to be one of the most effective inducers of neuroblastoma (NB) cell differentiation. Since increasing evidence indicates that expression of MHC class-I and class-II antigens by tumour cells is important for immunorecognition and cell targeting, we tested whether induction of NB cell differentiation by IFN-gamma is followed by expression of HLA class-I and class-II molecules. LAN-5 human NB cell line completely lacks HLA class-I antigens. Their expression was induced in a dose-dependent manner by IFN-gamma. HLA class-II molecules are also absent on LAN-5 cells, but only DP antigens were dose-dependently induced by IFN-gamma, while DR and DQ molecules were unaffected by the treatment. To confirm and extend the immunological data to all the class-II molecules, we performed Northern blot analysis, observing that DP alpha mRNA was induced in a dose- and time-dependent manner. DO beta and DZ alpha genes were also induced peaking after 3 days of IFN-gamma treatment. DR beta and DQ beta genes, which were not induced by IFN-gamma, gave a normal pattern of enzyme restriction by Southern blot. To get an insight into the regulation of HLA class-II gene expression in the neuronal model, we measured the decline of the steady-state HLA class-II mRNA. DO beta mRNA rapidly returned to baseline level after removing IFN-gamma, while the decay rates of DP alpha and DZ alpha mRNA were very slow. This might indicate different regulation at the post-transcriptional level for DO beta mRNA with respect to DP alpha and DZ alpha mRNA. To strengthen these findings we evaluated the half-lives of the mRNA after IFN-gamma induction by means of actinomycin D treatment. HLA-DO beta mRNA had a shorter half-life, while DZ alpha and DP alpha had a longer decay rate. Finally, we report that treatment of LAN-5 cells with cycloheximide did not alter the rate of transcription of the HLA-DP alpha gene, suggesting that no protein factor(s) is/are needed to maintain DP alpha gene expression.

Diastolic perfusion time at ischemic threshold in patients with stress-induced ischemia.

BACKGROUND: To evaluate the relevance of diastolic perfusion time on the mechanisms underlying stress-induced ischemia, 16 patients with coronary artery disease and seven patients with syndrome X underwent five randomized stress tests (upright and supine exercise with and without therapy, transesophageal atrial pacing). METHODS AND RESULTS: Exercise duration Time to 0.1 mV ST segment depression, heart rate, rate-pressure product, and diastolic perfusion time were evaluated for each patient during stress tests. In both groups, variability coefficients of the above-mentioned parameters were not different at rest. At ischemic threshold (0.1 mV ST segment depression) in patients with coronary artery disease, the variability coefficient of exercise duration (40.1 +/- 22.2) was significantly higher (p less than 0.0001) than those of heart rate (12.8 +/- 2.9), rate-pressure product (14.8 +/- 3.3), and diastolic perfusion time (0.39 +/- 0.1). The variability coefficient of diastolic perfusion time was also significantly (p less than 0.0001) lower than those of heart rate and rate-pressure product. Similarly, the variability coefficient of diastolic perfusion time (0.44 +/- 0.1) in syndrome X patients was significantly lower (p less than 0.0001) than those of exercise duration (28.2 +/- 9.4), heart rate (12 +/- 1.4), and rate-pressure product (14.6 +/- 1.3). CONCLUSIONS: Fixed diastolic perfusion time at ischemic threshold, despite different kinds of stress tests and variability of heart rate and rate-pressure product, indicates the relevant role of diastolic perfusion time in determining myocardial ischemia.