Cytokine-enhanced vaccine and suicide gene therapy as surgery adjuvant treatments for spontaneous canine melanoma: 9 years of follow-up.

We present here the updated results after 9 years of the beginning of a trial on canine patients with malignant melanoma. This surgery adjuvant approach combined local suicide gene therapy with a subcutaneous vaccine composed by tumor cells extracts and xenogeneic cells producing human interleukin-2 and granulocyte-macrophage colony-stimulating factor. Toxicity was absent or minimal in all patients (0≤VCOG-CTCAE grade≤1). With respect to surgery-treated controls (ST), the complete surgery (CS) arm of this combined treatment (CT) significantly increased the fraction of local disease-free patients from 13 to 81% and distant metastases free from 32 to 84%. Even though less effective than the CS arm, the partial surgery (PS) arm of this CT was significantly better controlling the disease than only surgery (14% while PS-ST: 0%, P<0.01 and CS-ST: 5%, P<0.05). In addition, CT produced a significant sevenfold (CS) and threefold (PS) increase in overall survival. The CS-CT arm significantly improved both CS-ST metastasis-free- and melanoma overall survival from 99 days (respective ranges: 11-563 and 10-568) to >2848 days (81-2848 and 35-2848). Thus, more of 50% of our CT patients died of melanoma unrelated causes, transforming a lethal disease into a chronic one. Finally, surgery adjuvant CT delayed or prevented post-surgical recurrence and distant metastasis, significantly improved disease-free and overall survival maintaining the quality of life. Long-term safety and efficacy of this treatment are supported by the high number of CT patients (283) and extensive follow-up (>9 years). The successful clinical outcome encourages the further translation of similar approaches to human gene therapy trials.

Interferon-ß lipofection I. Increased efficacy of chemotherapeutic drugs on human tumor cells derived monolayers and spheroids.

We evaluated the effect of hIFNß gene transfer alone or in combination with different antineoplastic drugs commonly used in cancer treatment. Five human tumor-derived cell lines were cultured as monolayers and spheroids. Four cell lines (Ewing sarcomas EW7 and COH, melanoma M8 and mammary carcinoma MCF-7) were sensitive to hIFNß gene lipofection. Although this effect appeared in both culture configurations, spheroids showed a relative multicellular resistance (insensitive colon carcinoma HT-29 excluded). EW7 and M8 hIFNß-expressing cells were exposed to different concentrations of bleomycin, bortezomib, carboplatin, doxorubicin, etoposide, methotrexate, paclitaxel and vincristine in both configuration models. In chemotherapy-sensitive EW7 monolayers, the combination of hIFNß gene and antineoplastic drugs displayed only additive or counteractive (methotrexate) effects, suggesting that cytotoxic mechanisms triggered by hIFNß gene lipofection could be saturating the signaling pathways. Conversely, in chemotherapy-resistant EW7 spheroids or M8 cells, the combination of hIFNß with drugs that mainly operate at the genotoxic level (doxorubicin, methotrexate and paclitaxel) presented only additive effects. However, drugs that also increase pro-oxidant species can complement the antitumor efficacy of the hIFNß gene and clearly caused potentiated effects (bleomycin, bortezomib, carboplatin, etoposide and vincristine). The great bystander effect induced by hIFNß gene lipofection could be among the main causes of its effectiveness, because only 1 or 2% of EW7 or M8 hIFNß-expressing cells killed more than 60 or 80% of cell population, respectively.

Interferon-ß lipofection II. Mechanisms involved in cell death and bystander effect induced by cationic lipid-mediated interferon-ß gene transfer to human tumor cells.

We evaluated the cytotoxic effects (apoptosis, necrosis and early senescence) of human interferon-ß (hIFNß) gene lipofection. The cytotoxicity of hIFNß gene lipofection resulted equivalent to that of the corresponding addition of the recombinant protein (rhIFNß) on human tumor cell lines derived from Ewing's sarcoma (EW7 and COH) and colon (HT-29) carcinomas. However, it was stronger than rhIFNß on melanoma (M8) and breast adenocarcinoma (MCF7). To reveal the mechanisms involved in these differences, we compared the effects of hIFNß gene and rhIFNß protein on EW7 and M8 (sensitive and resistant to rhIFNß protein, respectively). Lipofection with hIFNß gene caused a mitochondrial potential decrease simultaneous with an increase of oxidative stress in both cell lines. However, rhIFNß protein displayed the same pattern of response only in EW7-sensitive cell line. The great bystander effect of the hIFNß gene lipofection, involving the production of reactive oxygen species, would be among the main causes of its success. In EW7, this effect killed >60% of EW7 cell population, even though only 1% of cells were expressing the transgene. As hIFNß gene was effective even in the rhIFNß protein-resistant M8 cell line and in a way not limited by low lipofection efficiency, these results strongly support the clinical potential of this approach.

Suicide gene therapy on spontaneous canine melanoma: correlations between in vivo tumors and their derived multicell spheroids in vitro.

To validate the use of multicellular spheroids to predict the efficacy of herpes simplex thymidine kinase/ganciclovir (HSVtk/GCV) suicide gene therapy in the respective in vivo tumors, we established and characterized 15 melanoma-derived cell lines from surgically excised melanoma tumors. Three HSVtk-lipofected cell lines were not sensitive to GCV in any culture configuration, other five displayed similar sensitivity as monolayers or spheroids, and only one resulted more sensitive when grown as spheroids. Other six cell lines manifested a relative multicellular resistance (MCR) phenotype growing as spheroids, compared with the same cells growing as monolayers. The reverse correlation between the MCR and the monolayers survival to HSVtk/GCV suggests that one of the main causes of MCR would be the rapid cell repopulation after suicide gene treatment. The high correlation of MCR with the spheroids radial growth and with the mitotic index of the respective originary tumors supported this re-growth involvement. A remarkable finding was the high correlation in HSVtk/GCV sensitivity between in vivo tumor and the corresponding derived cell lines growing as spheroids (R(2) = 0.85). This strongly encourages the implementation of spheroids as highly realistic experimental model for optimizing and predicting the in vivo response of the respective tumors to therapeutic strategies.

Suicide gene and cytokines combined nonviral gene therapy for spontaneous canine melanoma.

Canine spontaneous melanoma is a highly aggressive tumor resistant to current therapies. We evaluated the safety, efficacy and antitumor effects of direct intratumor injections of lipoplexes encoding herpes simplex thymidine kinase coadministrated with ganciclovir, and irradiated transgenic xenogeneic cells secreting 20-30 mug day(-1) of human granulocyte-macrophage colony-stimulating factor and interleukin-2. Toxicity was minimal or absent in all patients. This combined treatment (CT) induced tumor regression and a pronounced immune cell infiltration. The objective responses (47%: 21/45) averaged 80% of tumor mass loss. Local CT also induced systemic antitumor response evidenced by complete remission of one pulmonary metastasis and by the significantly higher percentage of metastasis-free patients (76: 34/45)) until the study ending compared to untreated (UC: 29%, 5/17), surgery-treated (CX: 48%, 11/23) or suicide gene-treated controls (SG: 56%, 9/16) (Fisher's exact test). CT significantly improved median survival time: 160 (57-509) days compared to UC (69 (10-169)), CX (82 (43-216)) or SG (94 (46-159)). CT also increased (P<0.00001, Kaplan-Meier analysis) metastasis-free survival: >509 (57-509) days with respect to UC: 41 (10-169), CX: 133 (43-216) and SG: >159 (41-159). Therefore, CT controlled tumor growth by delaying or preventing distant metastasis, thereby significantly extending survival and recovering the quality of life.

Cytokine-enhanced vaccine and suicide gene therapy as surgery adjuvant treatments for spontaneous canine melanoma.

We evaluated the safety, efficacy and anti-tumor effects of a surgery adjuvant treatment on canine patients with malignant melanoma. This approach combined suicide gene therapy with a subcutaneous vaccine composed by formolized tumor cells and irradiated xenogeneic cells producing human interleukin-2 and granulocyte-macrophage colony-stimulating factor. The post-surgical margin of the cavity was infiltrated with lipid-complexed thymidine kinase suicide gene coadministrated with ganciclovir. Toxicity was minimal or absent in all patients. With respect to surgery-treated controls (SC), this combined treatment (CT) significantly increased the fraction of patients local disease-free from 6 to 58% and distant metastases-free from 43 to 78% (Fisher's Exact test). In addition, CT significantly improved both SC overall 78 (23-540) and metastasis-free survival 112 (0-467) days to more than 1312 days (respective ranges: 43-1312 and 0-1312) (Kaplan-Meier analysis). In those patients subjected to partial surgery or presenting local recurrence, the efficacy of CT was verified by a 49% of objective responses that averaged 85% of tumor mass loss, while 22% displayed tumor progression as 94% of SC did. Therefore, surgery adjuvant CT controlled tumor growth, delaying or preventing post-surgical recurrence and distant metastasis, significantly extending survival and recovering the quality of life.

Intracellular melatonin distribution in cultured cell lines.

A specific antibody combined with a fluorescein-labeled immunoglobulin was used to investigate the topographic distribution of melatonin in a variety of cells of different origins. Positive identification of both nuclear and cytosolic melatonin was confirmed in all the tested cells: Swiss 3T3 mouse fibroblasts, BCG1 bovine granulosa, NB41A3 mouse neuroblastoma, F9 mouse teratocarcinoma, MDCK normal canine kidney derived and human HeLa cell lines, as well as in human peripheral blood mononuclear leukocytes and rat splenic cells. In 3T3 mouse fibroblasts melatonin immunofluorescence partially colocalized with actin and serotonin immunostaining, but not with tubulin or actin stress fibers. Several distinct patterns of subcellular melatonin distribution, different from the bromodeoxyuridine-labeled replication profiles, have been discerned throughout the cell cycle of synchronized 3T3 cells. In addition, synchronized 3T3 mouse fibroblasts cultured in the presence of 10(-3) M melatonin progressed more slowly through the cell cycle than control cells. These results suggest that melatonin may interact directly with nuclear and cytoskeletal structures probably affecting different cell functions such as cell cycle control, subcellular organization, and genome stability.

Cultured peripheral blood mononuclear leukocytes from anorexia nervosa patients are refractory to visible light.

Cultured human peripheral blood mononuclear leukocytes [PBML] from patients with anorexia nervosa [AN] did not respond to light stimulation as PBML of normal controls [NC] did. During winter, visible light increased [3H]thymidine incorporation into DNA of NC-PBML stimulated with phytohemagglutinin [PHA]. This effect was enhanced by 10(-7) M melatonin. PHA-stimulated DNA synthesis of PBML from AN patients failed to respond to photic stimulation during winter, and their proliferative response to melatonin was significantly blunted. In vitro photic stimulation of NC-PBML reduced melatonin while increasing both serotonin and 5-hydroxyindole 3-acetic acid [HIAA] production in both basal and PHA-stimulated conditions. In contrast AN-PBML, that in darkness enhanced the oxidative deamination of serotonin into HIAA more than NC-PBML, did not switch their indole metabolism in response to light. Light did not inhibit the binding of both melatonin and serotonin to AN-PBML as occurred in NC-PBML. The present data suggest that AN-PBML do not respond to light in vitro, because of a failure in the regulation of serotonin and melatonin metabolism.

Sensitivity of human peripheral blood mononuclear leukocytes to visible light.

Overnight light exposure of cultured human peripheral blood mononuclear leukocytes [PBML], significantly increased basal [3H]thymidine incorporation and upon stimulation with phytohemagglutinin [PHA]. Melatonin (10(-9) to 10(-5) M) enhanced the light-induced increase of [3H]thymidine incorporation, while serotonin (10(-9) to 10(-7) M) stimulated [3H]thymidine incorporation in the dark. The wavelengths responsible of this effect were restricted to the blue-green zone of the spectrum. The stimulatory effect of visible light on PHA-induced DNA replication had a circannual rhythm, being maximal during winter. In winter, white light also reduced melatonin and serotonin binding to PBML membranes and switched the PBML indole metabolism towards serotonin and 5-hydroxy-indole-acetic acid [HIAA] synthesis, with a concomitant decrease of melatonin production.

DNA supercoiling and thermal regulation of unsaturated fatty acid synthesis in Bacillus subtilis.

Bacillus subtilis growing at 37 degrees C synthesizes, almost exclusively, saturated fatty acids. However, when a culture growing at 37 degrees C is transferred to 20 degrees C, the synthesis of unsaturated fatty acids is induced. The addition of the DNA gyrase inhibitor novobiocin specifically prevented the induction of unsaturated fatty acid synthesis at 20 degrees C. Furthermore, it was determined that plasmid DNA isolated from cells growing at 20 degrees C was significantly more negatively supercoiled than the equivalent DNA isolated from cells growing at 37 degrees C. The overall results agree with the hypothesis that an increase in DNA supercoiling associated with a temperature downshift could regulate the unsaturated fatty acids synthesis in B. subtilis.

Interactions of Drosophila DNA topoisomerase II with left-handed Z-DNA in supercoiled minicircles.

The native form of Drosophila melanogaster DNA topoisomerase II was purified from Schneider's S3 tissue culture cells and studied with two supercoiled minicircle preparations, mini and mini-CG, 354 bp and 370 bp in length, respectively. Mini-CG contains a d(CG)7 insert which assumes a left-handed Z-DNA conformation in negative supercoiled topoisomers with a negative linking number difference - delta Lk greater than or equal to 2. The interactions of topoisomerase II with topoisomer families of mini and mini-CG were studied by band-shift gel electrophoresis in which the individual topoisomers and their discrete or aggregated protein complexes were resolved. A monoclonal anti-Z-DNA IgG antibody (23B6) bound and aggregated only mini-CG, thereby confirming the presence of Z-DNA. Topoisomerase II bound and relaxed mini-CG more readily than mini. In both cases, there was a preference for more highly negatively supercoiled topoisomers. The topoisomerase II inhibitor VM-26 induced the formation of stable covalent DNA-protein intermediates. In addition, the non-hydrolyzable GTP analogue GTP gamma S inhibited the binding and relaxation activities. Experiments to detect topoisomerase cleavage sites failed to elicit specific loci on either minicircle preparation. We conclude that Drosophila topoisomerase II is able to bind and process small minicircles with lengths as short as 360 bp and negative superhelix densities, - sigma, which can exceed 0.1. Furthermore, the enzyme has a preferential affinity for topoisomers containing Z-DNA segments and relaxes these molecules, presumably by cleavage external to the inserts. Thus, a potentially functional relationship between topoisomerase II, an enzyme regulating the topological state of DNA-chromatin in vivo, and left-handed Z-DNA, a conformation stabilized by negative supercoiling, has been established.

Single strand binding protein specific for the polyoma early-coding strand of PEA1 (AP1) regulatory sequence.

We have shown that nuclear and cytosolic proteins from embryonal carcinoma F9 cells are able to bind to the early-coding strand of polyoma enhancer A domain. As demonstrated by mobility shift specific competition experiments, DNase I footprinting, depurination and depyrimidation interference, and proteolytic clipping performed with single stranded oligonucleotides, some of these proteins bind specifically to the early-coding PEA1 (AP1) motif. In addition, 'Southwestern' analysis has made possible the identification of a 46 KD nuclear protein that binds to this sequence. These cellular proteins did not bind to the complementary single strand as demonstrated by mobility shift analysis, nor did they bind to RNA synthesized in vitro by using the complementary strand as template. They were also shown to be different from their corresponding double strand binding factors. This new dimension in the functional flexibility and complexity of the polyoma enhancer suggests new properties of the classic regulating sequences that could provide additional modulation of regulating activities.

A DNA topoisomerase I inhibitor blocks the differentiation of rat granulosa cells induced by follicle-stimulating hormone.

Follicle-stimulating hormone (FSH), acting through a cycle AMP-mediated mechanism, promotes differentiation of rat granulosa cells cultured in a defined medium. Camptothecin, a DNA topoisomerase I blocker, inhibited the increase in progesterone and oestradiol production stimulated by FSH. This effect was not due to non-specific inhibition of protein synthesis, as shown by measurement of [35S]methionine incorporation. A transient increase in DNA topoisomerase I activity was observed after 24 h of culture in the presence of FSH or dibutyryl cyclic AMP. Our results are consistent with a key role for DNA topoisomerase I in the modulation of gene expression by FSH in rat granulosa cells.

Requirement of DNA topoisomerases for in vitro chromatin assembly by 3T6 mouse cell extracts.

Nuclear extracts of 3T6 mouse cells were able to assemble in vitro minichromosomes which displayed a 150-bp periodicity. Activities of both DNA topoisomerases I and II were detected in these extracts. When a supercoiled pUC DNA was added, it was first relaxed in less than 3 min, then slowly supercoiled again in 1-4 h. Both reactions occurred either in the absence or the presence of added Mg2+ and/or ATP, they were not blocked by DNA topoisomerase II inhibitors and they were inhibited by an antiserum against DNA topoisomerase I and by camptothecin. These findings led us to propose that, under our in vitro assay conditions, chromatin assembly is mainly carried out by a DNA topoisomerase I.

A novel stimulator of protein phosphorylation in Neurospora crassa.

An endogenous thermostable activator of Protein kinase III (PKIII) was purified from 100,000 X g supernatants of Neurospora crassa mycelial extracts. This 38,000 dalton polypeptide, clearly separable from calmodulin on P-60 gel filtration, specifically stimulated N. crassa PKIII activity on casein or phosvitin "in vitro" phosphorylation. The factor was only present in the initial growth phase of the fungus. The mechanism of PKIII activation and its possible regulatory role are discussed.

In vitro transcription by Xenopus oocytes RNA polymerase III requires a DNA topoisomerase II activity.

Extracts of Xenopus laevis oocytes are able to assemble minichromosomes in vitro when they are supplemented with ATP and Mg2+. We have followed the time course of in vitro DNA supercoiling and transcription of polyoma virus closed circular DNA. The transcriptional activity increased with the assembly time of chromatin in the presence of both ATP and Mg2+, but only residual activity was detected in the absence of either of them. We also found that polyoma DNA as well as 5S RNA gene transcription were carried out mainly by an RNA polymerase III. On the other hand, polyoma RNA and 5S RNA synthesis were inhibited by novobiocin in a way that suggested the requirement of a DNA topoisomerase II or DNA gyrase activity for the initiation of transcription.

Characterization of Neurospora crassa cyclic AMP phosphodiesterase activated by calmodulin.

Activation of cyclic AMP phosphodiesterase I by brain or Neurospora calmodulin was studied. The stimulation required micromolar concentrations of Ca2+, and it was observed at cyclic AMP concentrations between 0.1 and 500 microM. Activation was blocked by EDTA and some neuroleptic drugs such as chlorpromazine and fluphenazine. These drugs inhibit the elongation of N. crassa wild-type aerial hyphae. These results reinforce the evidence towards the recognition of Ca2+-calmodulin as one of the systems controlling cyclic nucleotide concentrations in Neurospora.

Chromatin assembly in Xenopus oocytes: in vitro studies.

We describe and characterize a complex reaction that catalyzes DNA supercoiling and chromatin assembly in vitro. A Xenopus oocyte extract supplemented with ATP and Mg++ converts DNA circles into minichromosomes that display a native, 200 bp periodicity. When supercoiled DNA is added to this extract it undergoes a time-dependent series of topological changes, which precisely mimic those found when the DNA is microinjected into oocytes. As judged by the conformation of the subsequently deproteinized DNA, the supercoiled DNA is first relaxed, in a reaction that takes 4 min, and then it is resupercoiled in a slower process that takes 4 hr. The relaxation is partially inhibited by EDTA, to an extent that suggests that that it is catalyzed by a type I DNA topoisomerase. The resupercoiling , on the other hand, requires ATP and Mg++, is completely inhibited by EDTA, and is inhibited by novobiocin in a manner that suggests it is catalyzed by a type II DNA topoisomerase. These findings, and the ones reported in the preceding paper ( Ryoji and Worcel , 1984), lead us to propose that chromatin assembly is an active, ATP-driven process.

Osmium tetroxide: a new probe for site-specific distortions in supercoiled DNAs.

Supercoiled plasmids Col E1 and cDm 506 (a Col E1 derivative carrying the D. melanogaster histone gene repeat) were treated with OsO4 in presence of pyridine and the reaction products were analyzed using different approaches. Gel electrophoresis showed that OsO4 binding to supercoiled DNA induced its relaxation without nicking. The amount of osmium bound to DNA (as determined electrochemically) increased with the extent of DNA relaxation. As a result of osmium modification of supercoiled cDm 506, a single denaturation "bubble" was observed in the electron microscope. Mapping of the osmium binding site by S1 nuclease cleavage followed by restriction enzyme digestion has revealed one major site in the intergenic spacer between the H1 and H3 histone genes of D. melanogaster. This site differs from the site cleaved by S1 nuclease in supercoiled DNA in the absence of osmium.