Inhibition Of Colony Growth Research Articles

Antioxidants protect the yeast Saccharomyces cerevisiae against hypertonic stress

Yeast (Saccharomyces cerevisiae) mutants lacking CuZnSOD have been reported to be hypersensitive to hypertonic media and to show increased oxidative damage. This study demonstrates that hypertonic medium (containing 0.8 M NaCl) increases the generation of superoxide and other reactive species in yeast cells. Other sequelae of exposure to hypertonic medium include oxidation of cellular low-molecular weight thiols and decrease in total antioxidant capacity of cellular extracts. Δsod1 mutant is more sensitive than a wild-type strain to colony growth inhibition on a hypertonic medium. Anaerobic conditions, ascorbate, glutathione, cysteine and dithiothreitol are able to ameliorate this growth inhibition but a range of other antioxidants does not protect. The protective ability of the antioxidants does not correlate with the rate of their reactions with superoxide but seems to be conditioned by low redox potential for one-electron oxidation of free radicals of the antioxidants. It suggests that repair of low-redox potential targets rather than prevention of their damage by superoxide is important in the antioxidant protection against oxidative stress induced by hypertonic conditions.

Pim-1 is up-regulated by constitutively activated FLT3 and plays a role in FLT3-mediated cell survival

AbstractConstitutively activating internal tandem duplication (ITD) mutations of the receptor tyrosine kinase FLT3 (Fms-like tyrosine kinase 3) play an important role in leukemogenesis, and their presence is associated with poor prognosis in acute myeloid leukemia (AML). To better understand FLT3 signaling in leukemogenesis, we have examined the changes in gene expression induced by FLT3/ITD or constitutively activated wild-type FLT3 expression. Microarrays were used with RNA harvested before and after inhibition of FLT3 signaling. Pim-1 was found to be one of the most significantly down-regulated genes upon FLT3 inhibition. Pim-1 is a proto-oncogene and is known to be up-regulated by signal transducer and activator of transcription 5 (STAT5), which itself is a downstream target of FLT3 signaling. Quantitative polymerase chain reaction (QPCR) confirmed the microarray results and demonstrated approximately 10-fold decreases in Pim-1 expression in response to FLT3 inhibition. Pim-1 protein also decreased rapidly in parallel with decreasing autophosphorylation activity of FLT3. Enforced expression of either the 44-kDa or 33-kDa Pim-1 isotypes resulted in increased resistance to FLT3 inhibition-mediated cytotoxicity and apoptosis. In contrast, expression of a dominant-negative Pim-1 construct accelerated cytotoxicity in response to FLT3 inhibition and inhibited colony growth of FLT3/ITD-transformed BaF3 cells. These findings demonstrate that constitutively activated FLT3 signaling up-regulates Pim-1 expression in leukemia cells. This up-regulation contributes to the proliferative and antiapoptotic pathways induced by FLT3 signaling. (Blood. 2005;105: 1759-1767)

Potential Link between the NIMA Mitotic Kinase and Nuclear Membrane Fission during Mitotic Exit inAspergillus nidulans

We have isolated TINC as a NIMA-interacting protein by using the yeast two-hybrid system and have confirmed that TINC interacts with NIMA in Aspergillus nidulans. The TINC-NIMA interaction is stabilized in the absence of phosphatase inhibitors and in the presence of kinase-inactive NIMA, suggesting that the interaction is enhanced when NIMA is not fully activated. TINC is a cytoplasmic protein. TINC homologues and a TINC-like protein (A. nidulans HETC) are conserved in other filamentous fungi. Neither deletion of tinC nor deletion of both tinC and A. nidulans hetC is lethal, but deletion of tinC does produce cold sensitivity as well as osmotic sensitivity. Expression of an amino-terminal-truncated form of TINC (DeltaN-TINC) inhibits colony growth in Aspergillus and localizes to membrane-like structures within the cell. Examination of cell cycle progression in these cells reveals that they progress through multiple defective mitoses. Many cells contain large polyploid single nuclei, while some appear to have separated masses of DNA. Examination of the nuclear envelopes of cells containing more than one DNA mass reveals that both DNA masses are contained within a single nuclear envelope, indicating that nuclear membrane fission is defective. The ability of these cells to separate DNA segregation from nuclear membrane fission suggests that this coordination is normally a regulated process in A. nidulans. Additional experiments demonstrate that expression of DeltaN-TINC results in premature NIMA disappearance in mitotic samples. We propose that TINC's interaction with NIMA and the cell cycle defects produced by DeltaN-TINC expression suggest possible roles for TINC and NIMA during nuclear membrane fission.

The IKK Inhibitor PS1145 Allows to Overcome Imatinib Resistance.

Since a number of CML patients are resistant to Imatinib, additional molecular defects should be identified and targeted to improve the therapeutic strategies. A constitutive NF-kB activity has been demonstrated in several hematologic malignancies, therefore NF-kB blocking approaches have been introduced as antineoplastic strategies. The phosphorilation of IKB by IKK leads to IKB degradation by the proteasome, so freeing NF-kB to enter the nucleus and activate transcription. IKK may therefore represent an attractive target for molecular therapies. The aim of the study was to evaluate the effects of the IKK inhibitor PS1145 (Millenium) in CML cell lines and patients sensitive and resistant to Imatinib. K562 and KCl cells both sensitive (s) and resistant (r) to Imatinib and the BM cells collected from 13 CML patients were incubated with PS1145 10 μM, with Imatinib 1 μM and with the combination of the two drugs for 24 and 48 hrs. 11 out of 13 patients were in chronic phase, 1 in accelerate phase and 1 in blastic phase. 3 out of 11 were cytogenetic resistant, one was also hematologically resistant. The NF-kB activity was evaluated by ELISA method, the proliferation rate by MTT assay, apoptosis by FACS. Immunofluorescence was use to identify the NF-kB localization. Moreover colony growth was evaluated in control and treated samples. PS1145 was able to reduce the DNA binding activity of NF-kB of 90% and 85% respect to the control in K562 and KCL cells.This was confirmed by the prevailing cytoplasmatic localization of NF-kB after PS1145 incubation. In K562s and KCls treated with Imatinib, we observed a reduction of proliferation of 48% and 37% respectively. By contrast no effect on proliferation was observed in K562r and KClr. The incubation with PS1145 inhibited the proliferation of 38% and 15% in K562s and KCls, and of 22% and 28% in K562r and KClr. Interestingly in resistant cell lines the association of Imatinib and PS1145 induced a marked inhibition of proliferation of 87% in K562r and 82% in KClr. Similarly, PS1145 inhibited BM cells proliferation of 30% (range 11% to 65%). Imatinib incubation of BM cells from sensitive patients reduced the proliferation rate of 41% (range 29%–55%) but no effects were observed in resistant patients. In the three resistant patients the incubation with both compounds resulted in an increased block of proliferation respect to PS1145 alone with an inhibition of 42%, 49% and 58% after 24 hrs and 70%, 77% and 79% after 48 hrs. Imatinib plus PS1145 induced a significant increase of apoptosis from 7% to 69% in K562r and from 9 % to 71% in KClr. In agreement this association induced 48%, 52% and 39% of apoptotic cells in BM from the three resistant patients and a colony growth inhibition of 86%. Our data clearly demonstrated that the IKK inhibitor PS1145 is able to induce growth arrest and apoptosis in CML cell lines and BM cells. This effect is more sound in Imatinib resistant cells treated with the association of Imatinib and PS1145. Although the exact mechanism of action of PS1145 in resistant cells is at present a pure speculation, these data may suggest an intriguing mechanism to induce apoptosis in imatinib resistant cells based on imatinib resistance itself. The combination of Imatinib and the IKK inhibitor could therefore represent a valid approach for the treatment of CML patients resistant to Imatinib therapy.

In Vitro Exposure of Bacteria to Antimicrobial Impregnated-Central Venous Catheters Does Not Directly Lead to the Emergence of Antimicrobial Resistance

Use of central venous catheters (CVCs) impregnated with minocycline and rifampin reduces the density of bacterial growth on catheters and decreases the incidence of catheter-related bloodstream infections. Questions have been raised over the possibility that the use of these catheters will lead to the emergence of antibiotic-resistant organisms. In this study, we sought to determine if in vitro exposure of four test organisms to catheter segments impregnated with minocycline and rifampin would lead to the development of antibiotic resistance. Catheter segments (1.0 cm) were placed on the surface of agar plates previously inoculated with bacterial suspensions, such that a subconfluent lawn of colony growth would be apparent after 24 h incubation at 35 degrees C in air. Test organisms included American Type Culture Collection strains of Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa. Zones of inhibition of colony growth surrounding catheters were measured at 24-h intervals up to 7 days (two catheter segments per test). Colonies on agar surfaces located at varying distances from catheter segments were examined for minocycline and rifampin resistance following various periods of exposure (six catheter segments per test). In addition, selected colonies were subsequently exposed to minocycline and rifampin in broth and examined for selection of minocycline and rifampin resistance (> 28 colonies per selection test). Inhibitory zones of 14 to 47 mm were observed with S aureus, S epidermidis, E faecalis, and E coli. Growth of P aeruginosa was not inhibited by CVC segments. Testing of colonies of the first four organisms at various distances from CVC segments after varying periods of exposure revealed only a single instance of the emergence of resistance (eg, S aureus vs rifampin). Recovery of resistant clones was enhanced with minocycline and rifampin broth selection; however, a direct link between CVC exposure and the emergence of resistance was not established. Our in vitro data suggest that the exposure of Gram-positive cocci to either rifampin or minocycline can lead to the development of resistance. However, exposure of bacteria to these antibiotics in combination does not directly lead to resistance. Clinical investigations will be required to determine the true risk and implications of the development of resistance.

Inhibition of the benzo[a]pyrene-induced toxicity by allyl sulfides in human epidermal keratinocytes

Diallyl sulfide (DAS) and diallyl disulfide (DADS) at 25 microg ml(-1)decreased the benzo[a]pyrene (B[a]P)-induced colony growth inhibition of human epidermal keratinocytes. DAS and DADS decreased B[a]P-DNA and B[a]P-protein adducts by 65% and 49-55%, respectively. The B[a]P-induced ethoxyresorufin O-deethylase activity, a marker enzyme for cytochrome P450 1, was decreased from 3 to 1.7-1.9 nmol min(-1) mg(-1) microsomal protein by DAS and DADS treatments. The activity of glutathione S-transferase, a detoxifying enzyme for B[a]P, but was decreased by DADS, but was unaffected by DAS.

Ascorbic acid appears to increase the in vitro specificity and sensitivity of arsenic trioxide to the inhibition of primary myeloid colonies from patients with myelofibrosis with myeloid metaplasia

6619 Background: Myelofibrosis with myeloid metaplasia (MMM) is a hematopoietic stem cell disorder with no effective medical therapy. We have reported the use of in vitro inhibition of myeloid colony growth as a screen for novel therapeutic agents in MMM (Leukemia 2003;17(5):849.). Arsenic trioxide (ATO) was of interest in MMM because it's mechanisms of action (pro-apoptotic and anti-angiogenic), and activity against other myeloid diseases (APL and MDS), suggest possible activity in MMM. Recently it has been shown that the depletion of glutathione (with ascorbic acid (AA)) may augment the efficacy of ATO(Leukemia 2003;17:931.). We used our in vitro model to evaluate ATO (with and without AA) in primary MMM cells. Methods: Peripheral blood mononuclear cells (PBMC) were isolated from 12 MMM patients and 15 controls (normal n=10, polycythemia vera n=3, essential thrombocythemia n=2). PBMC were plated on the Methocult (StemCell technologies) colony assay media. Serial dilutions of ATO (0–10 μM) (clinically sustained ATO concentrations of >1 μM (Blood 1997;89(9):3354) with and without AA (100 μM) were added. Myeloid colonies were assessed after 14 days. Results: ATO demonstrated activity inhibiting myeloid colony growth in the MMM patients with a mean IC50 of 1.05 μM. However, ATO demonstrated similar activity in suppressing normal myeloid colony growth with an IC50 of 1.25 μM. Intriguingly the depletion of glutathione (through the addition of AA) demonstrated an increase in specificity for the in vitro effects of the ATO. Specifically, the IC50 for MMM decreased to 0.7 μM with the addition of AA whereas the IC50 for normals increased to 1.97μM. Control patients with other myeloproliferative disorders demonstrated an increased sensitivity to ATO with the addition of AA, mirroring our results with MMM. Conclusions: ATO demonstrates in vitro activity against MMM myeloid progenitors in clinically achievable concentrations. The addition of AA to ATO may increase the activity and specificity of the agent. Pilot studies of combination ATO plus AA may be warranted in MMM. No significant financial relationships to disclose.

Ascorbic acid appears to increase the in vitro specificity and sensitivity of arsenic trioxide to the inhibition of primary myeloid colonies from patients with myelofibrosis with myeloid metaplasia

6619 Background: Myelofibrosis with myeloid metaplasia (MMM) is a hematopoietic stem cell disorder with no effective medical therapy. We have reported the use of in vitro inhibition of myeloid colony growth as a screen for novel therapeutic agents in MMM (Leukemia 2003;17(5):849.). Arsenic trioxide (ATO) was of interest in MMM because it's mechanisms of action (pro-apoptotic and anti-angiogenic), and activity against other myeloid diseases (APL and MDS), suggest possible activity in MMM. Recently it has been shown that the depletion of glutathione (with ascorbic acid (AA)) may augment the efficacy of ATO(Leukemia 2003;17:931.). We used our in vitro model to evaluate ATO (with and without AA) in primary MMM cells. Methods: Peripheral blood mononuclear cells (PBMC) were isolated from 12 MMM patients and 15 controls (normal n=10, polycythemia vera n=3, essential thrombocythemia n=2). PBMC were plated on the Methocult (StemCell technologies) colony assay media. Serial dilutions of ATO (0–10 μM) (clinically sustained ATO concentrations of >1 μM (Blood 1997;89(9):3354) with and without AA (100 μM) were added. Myeloid colonies were assessed after 14 days. Results: ATO demonstrated activity inhibiting myeloid colony growth in the MMM patients with a mean IC50 of 1.05 μM. However, ATO demonstrated similar activity in suppressing normal myeloid colony growth with an IC50 of 1.25 μM. Intriguingly the depletion of glutathione (through the addition of AA) demonstrated an increase in specificity for the in vitro effects of the ATO. Specifically, the IC50 for MMM decreased to 0.7 μM with the addition of AA whereas the IC50 for normals increased to 1.97μM. Control patients with other myeloproliferative disorders demonstrated an increased sensitivity to ATO with the addition of AA, mirroring our results with MMM. Conclusions: ATO demonstrates in vitro activity against MMM myeloid progenitors in clinically achievable concentrations. The addition of AA to ATO may increase the activity and specificity of the agent. Pilot studies of combination ATO plus AA may be warranted in MMM. No significant financial relationships to disclose.

Target: Interaction Domains

Two groups report the design of small molecule inhibitors of protein-protein interactions. Roehrl et al. used a fluorescence polarization assay to screen for molecules that would inhibit the recognition of nuclear factor of activated T cells (NFAT) by the phosphate calcineurin. Molecules were identified that decreased the fluorescence of a fluorescently tagged short peptide (VIVIT) that was known to interfere with the NFAT-calcineurin interaction. Molecules that completely displaced VIVIT were analyzed further. In addition to confirming the interaction of these molecules with calcineurin, the authors showed that one of them, INCA-6, blocked NFAT dephosphorylation, NFAT nuclear translocation, and stimulation of NFAT-dependent gene expression in cultured T cells. Calcineurin in lysates from cells exposed to INCA-6 retained catalytic activity. Gao et al. used a structure-based virtual screen to identify small molecules that would fit in the site on the Rac guanosine triphosphatase (GTPase) where its guanine exchange factor (GEF) interacts. Each GTPase has a selective GEF that stimulates it. Gao et al. identified a small molecule, NSC23766, that selectively blocked the interaction between Rac1 and its GEFs, TrioN and Tiam1, but did not affect the interaction between other GTPases and their GEFs. NSC23766 not only blocked GEF binding but also inhibited nucleotide exchange on Rac1. In cultured fibroblasts (NIH 3T3 cells), NSC23766 inhibited Rac activation, the formation of lamellipodia, and cell proliferation in response to growth factors. NSC23766 inhibited colony growth in soft agar caused by overexpression of Tiam1 and inhibited proliferation, anchorage-independent growth, and invasiveness of PC-3 prostate tumor cells. These two papers demonstrate the effectiveness of targeting protein interaction domains. M. H. A. Roehrl, S. Kang, J. Aramburu, G. Wagner, A. Rao, P. G. Hogan, Selective inhibition of calcineurin-NFAT signaling by blocking protein-protein interaction with small organic molecules. Proc. Natl. Acad. Sci. U.S.A. 101 , 7554-7559 (2004). [Abstract] [Full Text] Y. Gao, J. B. Dickerson, F. Guo, J. Zheng, Y. Zheng, Rational design and characterization of a Rac GTPase-specific small molecule inhibitor. Proc. Natl. Acad. Sci. U.S.A. 101 , 7618-7623 (2004). [Abstract] [Full Text]

Translocation products in acute myeloid leukemia activate the Wnt signaling pathway in hematopoietic cells.

The acute myeloid leukemia (AML)-associated translocation products AML1-ETO, PML-retinoic acid receptor alpha (RARalpha), and PLZF-RARalpha encode aberrant transcription factors. Several lines of evidence suggest similar pathogenetic mechanisms for these fusion proteins. We used high-density oligonucleotide arrays to identify shared target genes in inducibly transfected U937 cells expressing AML1-ETO, PML-RARalpha, or PLZF-RARalpha. All three fusion proteins significantly repressed the expression of 38 genes and induced the expression of 14 genes. Several of the regulated genes were associated with Wnt signaling. One of these, plakoglobin (gamma-catenin), was induced on the mRNA and protein level by all three fusion proteins. In addition, primary AML blasts carrying one of the fusion proteins significantly overexpressed plakoglobin. The plakoglobin promoter was cloned and shown to be induced by AML1-ETO, with promoter activation depending on the corepressor and histone deacetylase binding domains. The induction of plakoglobin by AML fusion proteins led to downstream signaling and transactivation of TCF- and LEF-dependent promoters, including the c-myc promoter, which was found to be bound by plakoglobin in vivo after AML1-ETO expression. beta-Catenin protein levels and TCF and LEF target genes such as c-myc and cyclin D1 were found to be induced by the fusion proteins. On the functional level, a dominant negative TCF inhibited colony growth of AML1-ETO-positive Kasumi cells, whereas plakoglobin transfection into myeloid 32D cells enhanced proliferation and clonal growth. Injection of plakoglobin-expressing 32D cells into syngeneic mice accelerated the development of leukemia. Transduction of plakoglobin into primitive murine hematopoietic progenitor cells preserved the immature phenotype during colony growth, suggesting enhanced self-renewal. These data provide evidence that activation of Wnt signaling is a common feature of several balanced translocations in AML.

Targeting the single-strand G-rich overhang of telomeres with PNA inhibits cell growth and induces apoptosis of human immortal cells

Telomeres are believed to stabilize chromosomes through several mechanisms that are dependent upon specific DNA–DNA and protein–DNA interactions. Telomeres are maintained by the enzyme telomerase. Telomerase activity, which is below detectable level in almost all types of diploid cells, is re-activated in most immortal and cancer cells. For this study, we designed peptide nucleic acid (PNA) oligonucleotides targeted to the telomeric G-rich strand, and tested their efficacy to reverse the immortality of transformed human fibroblasts. Anti-telomere PNAs, transfected into human fibroblasts along with a selectable marker, resulted in a significant reduction in colony size and elicited cell death by apoptosis. This PNA inhibitor does not inhibit telomerase activity in vitro, suggesting a distinct cellular mechanism from known PNA inhibitors. A combination of this class of PNA inhibitor with a PNA that does block telomerase activity resulted in nearly complete inhibition of colony growth, induction of apoptosis, and an apparent reduction in telomere length. Each effect was greater than that evoked by either agent alone, indicating enhanced efficacy for therapeutic approaches that target multiple, distinct mechanism of telomere maintenance.

In vivo erythroid recovery following paclitaxel injury: correlation between GATA-1, c-MYB, NF-E2, Epo receptor expressions, and apoptosis

Paclitaxel (Px) is a cancer chemotherapeutic agent that causes bone marrow (BM) cytotoxicity by microtubule stabilization and by modifications in the expression of several genes. Hematopoietic progenitors show severe alterations following Px injury. Erythropoietic recovery should be accompanied by changes in the expression of transcription factors such as c-MYB, GATA-1, NF-E2, Bcl-x L, and erythropoietin receptor (Epo-R). The aim of this work was to study the in vivo recovery of erythropoiesis and to correlate transcription factors, Bcl-x L, and Epo-R expressions to apoptosis and changes in proliferation of murine erythroid progenitors following a single dose of Px (29 mg/kg, i.p.). BM total and differential cellularities, apoptosis (TdT-mediated dUTP Nick-End Labeling [TUNEL] assay), clonogenic assays, and immunoblots for transcription factors, Epo-R, and Bcl-x L were performed each day for 5 days post-injury. Apoptosis (24 ± 0.81%, P < 0.01), inhibition of colony growth (burst-forming units-erythroid [BFU-E] and granulocyte-erythroid-macrophage [GEM]), and decrease in BM cellularities (28 ± 4.2% of control) were maximal at 24 h following Px. The highest apoptosis was concomitant with the lowest BM cellularities. Apoptosis returned to normal values (3.08 ± 0.61%) by day 3 post-Px. Up-regulation of c-MYB, GATA-1, Epo-R, and Bcl-x L expressions were observed between 24 and 48 h following Px. Correlations among c-MYB, GATA-1, Bcl-x L, and Epo-R were extremely significant. Maximal expression of NF-E2 was observed on day 3 concomitant with the rise (threefold) of early erythroid precursors (BFU-E). Thus, cells that survive injury seem to be stimulated to produce early (24–48 h) erythroid-related and antiapoptotic proteins. Therefore, the results suggest an in vivo interplay between specific transcription factors and Bcl-x L during progenitor cell survival and proliferation; mechanisms triggered to restore size and composition of the erythroid compartment.

Specific protein redirection as a transcriptional therapy approach for t(8;21) leukemia.

Important progress has been achieved in the knowledge about the pathogenesis of cancer. However, despite these advances, the therapeutic strategies are still limited. Leukemias are often characterized by specific balanced translocations, with the t(8;21) balanced translocation being the most frequent chromosomal aberration in acute myeloid leukemia (AML). This translocation produces the AML1-ETO fusion protein, which binds to AML1 target promoter sequences. Transcriptional repression of AML1-dependent genes by AML1-ETO and associated corepressors represents the pathogenetic mechanisms of t(8;21). Here, we show that targeting of AML1-ETO to essential, MYB-dependent gene promoters induces t(8;21)-restricted cell death. We constructed a chimeric protein that contained the MYB DNA-binding domain and the AML1-binding domain of myeloid Elf-1-like factor (MEF). This protein associated with AML1-ETO and directed the complex to MYB-responsive promoters in vitro and in vivo. In the presence of AML1-ETO, the chimeric protein repressed the activity of MYB-responsive promoters, rapidly induced apoptosis, and specifically inhibited colony growth. All these effects occurred only in AML1-ETO-positive cells, whereas no adverse effects were observed in cells not expressing AML1-ETO. Taken together, this study demonstrates that redirection of oncogenic proteins can be used as a strategy to dramatically influence their cellular effects, with the ultimate goal to design highly specific therapies for cancer.

Delayed addition of deoxycytidine protects normal CD34+ cells against cytotoxicity of gemcitabine without compromising its activity against human leukemic cells.

In phase I and II clinical trials, the deoxycytidine analogue 2',2' difluorodeoxycytidine (dFdC, gemcitabine) has shown promising antitumor activity in leukemia as well as in solid tumors. Preclinical and clinical studies of gemcitabine suggested that myelosuppression was the dose-limiting toxicity. The present investigations were designed to test the effect of continuously administered gemcitabine on the in vitro clonal growth of normal CD34(+) cells isolated from peripheral blood and the promyelocytic cell line, HL-60. For this purpose, CD34(+) and HL-60 cells were cultured in methylcellulose in the continuous presence of 0.1-16 nM of gemcitabine. The results show a dose-dependent inhibition of colony growth of normal as well as leukemic cells. However, HL-60 cells were up to 12-fold more sensitive towards gemcitabine than normal progenitors. For rescue experiments, the natural pyrimidine deoxycytidine (dCyd) was added to CD34(+) and HL-60 cells simultaneously or with delay. Coadministration of 1mM dCyd to separate cultures resulted in complete restoration of colony formation capacity of CD34(+) and HL-60 cells. Delayed addition of 1 mM dCyd after 48 and 72 hours recovered up to 90% and 40%, respectively, of stem cell proliferation, whereas HL-60 cells remained substantially inhibited (4.5% +/- 3.5% versus 0%). Delayed addition after 48 and 72 hours protected about 80% and 50%, respectively, of myelopoietic and erythropoetic colony formation, whereas colony formation obtained from HL-60 cells remained significantly inhibited (9.6% +/- 4.17% versus 0%). These in vitro data suggest that there is a marked difference in the susceptibility of leukemic and normal CD34(+) cells to gemcitabine and that delayed administration of dCyd may further reduce the bone marrow cytotoxicity of gemcitabine without impairing its antitumor effect.

Efeito do lodo de esgoto na indução de supressividade in vitro a Phytophthora nicotianae

Uma alternativa de manejo das doenças causadas por Phytophthora spp. é o uso de matéria orgânica. No presente trabalho foi avaliada a potencialidade do lodo de esgoto na indução de supressividade in vitro a P. nicotianae. O efeito do lodo de esgoto incorporado ao solo na sobrevivência de P. nicotianae foi avaliado mediante um experimento fatorial com dois fatores: doses de lodo de esgoto (0, 10, 20 e 40% p/p) e concentrações de inóculo [0, 10 ou 20 g de grãos de trigo (Triticum aestivum) colonizados kg-1]. Aos 21 dias, quando aumentaram as doses de lodo de esgoto, a sobrevivência de P. nicotianae e os pHs das misturas diminuíram, e as condutividades elétricas (CE) aumentaram. As correlações entre a CE e a sobrevivência do patógeno foram negativas e significativas (P&gt;0,05). Para estudar o efeito dos compostos químicos envolvidos na supressividade, foram obtidos extratos em água, H2SO4 2N e KOH 0,4N de misturas de areia – lodo de esgoto (20% p/p), e foram acrescentados ao meio de cultura e seu efeito avaliado no crescimento das colônias de P. nicotianae. O extrato ácido (H2SO4 2N) do tratamento com 20% de lodo de esgoto inibiu significativamente (P&gt;0,05) o crescimento da colônia do patógeno. O efeito biológico foi estudado mediante isolamento de microrganismos em meio de cultura e seleção por antagonismo. No bioensaio com plântulas de alfafa (Medicago sativa) destacaram-se os isolados F9.1 (Aspergillus sp.) e A12.1 (actinomiceto, não identificado); e no teste de culturas pareadas destacou-se um Trichoderma sp. e dois actinomicetos por antibiose, e um Trichoderma sp. e três Aspergillus sp. por hiperparasitismo.

Colony growth suppression by tumor suppressor genes.

Colony growth suppression by tumor suppressor genes.

The Influence of Imatinib Mesylate (STI571) used alone or in Combination with Purine Nucleoside Analogues on the Normal and Chronic Myelogenous Leukaemia Progenitor Cells In Vitro

Imatinib mesylate (STI571, Glivec), a signal transduction inhibitor used as a single agent demonstrates significant activity in patients with chronic myelogenous leukaemia (CML). Nevertheless, the interaction between STI571 and other antileukaemic drugs such as hydroxyurea, interferon α or cytarabine have also been investigated in order to further improve its effectiveness. In this study we have tried to answer the question if the combination of STI571 with purine nucleoside analogues (PNAs)- cladribine (2-CdA) and fludarabine (F-ara-A) intensifies the antiproliferative effect on granulocyte-macrophage progenitor cells (CFU-GM) from patients with CML as well as from normal persons. Our studies were based on the method of semisolid CFU-GM cultures in vitro. We added STI571 or PNAs singly to the culture, each of the drugs at three concentrations, as well as in combinations of the concentrations used. We showed that STI571 (0.5, 1.0 and 2.0 μM) used alone inhibited the colony growth of CML CFU-GM, as compared to CFU-GM derived from normal donors (p = 0.03; p = 0.0004; p = 0.0001). We also observed that STI571 used together with 2-CdA (5,10 and 20 nM) or F-ara-A (0.2, 0.4 and 0.8 μM) at all the combinations significantly inhibited the colony growth of CML CFU-GM, as compared either to the control or to STI571 used alone (p < 0.05). In addition, the differences between CML and normal CFU-GM colony growth inhibition after the use of the combination of the highest concentrations of STI571 either with 2-CdA or F-ara-A were statistically significant (p = 0.03 and p = 0.01, respectively). In conclusion, STI571 used together with both the PNAs had an additive effect on CML CFU-GM cells. However, further experimental and clinical studies concerning the usefulness of these combinations in the treatment of CML patients seem warranted.

The Influence of Imatinib Mesylate (STI571) used alone or in Combination with Purine Nucleoside Analogues on the Normal and Chronic Myelogenous Leukaemia Progenitor Cells In Vitro

Imatinib mesylate (STI571, Glivec), a signal transduction inhibitor used as a single agent demonstrates significant activity in patients with chronic myelogenous leukaemia (CML). Nevertheless, the interaction between STI571 and other antileukaemic drugs such as hydroxyurea, interferon α or cytarabine have also been investigated in order to further improve its effectiveness. In this study we have tried to answer the question if the combination of STI571 with purine nucleoside analogues (PNAs)- cladribine (2-CdA) and fludarabine (F-ara-A) intensifies the antiproliferative effect on granulocyte-macrophage progenitor cells (CFU-GM) from patients with CML as well as from normal persons. Our studies were based on the method of semisolid CFU-GM cultures in vitro. We added STI571 or PNAs singly to the culture, each of the drugs at three concentrations, as well as in combinations of the concentrations used. We showed that STI571 (0.5, 1.0 and 2.0 μM) used alone inhibited the colony growth of CML CFU-GM, as compared to CFU-GM derived from normal donors (p = 0.03; p = 0.0004; p = 0.0001). We also observed that STI571 used together with 2-CdA (5,10 and 20 nM) or F-ara-A (0.2, 0.4 and 0.8 μM) at all the combinations significantly inhibited the colony growth of CML CFU-GM, as compared either to the control or to STI571 used alone (p < 0.05). In addition, the differences between CML and normal CFU-GM colony growth inhibition after the use of the combination of the highest concentrations of STI571 either with 2-CdA or F-ara-A were statistically significant (p = 0.03 and p = 0.01, respectively). In conclusion, STI571 used together with both the PNAs had an additive effect on CML CFU-GM cells. However, further experimental and clinical studies concerning the usefulness of these combinations in the treatment of CML patients seem warranted.

On the cytotoxicity of some microbial volatile organic compounds as studied in the human lung cell line A549.

The cytotoxicity of 13 microbial volatile organic compounds (MVOC) was studied using a human lung carcinoma epithelial cell line A549 in a colony formation assay and two colorimetric assays: the microculture tetrazolium assay (MTT assay) and the cellular protein assay (methylene blue-MB assay). For comparison, two known cytotoxic substances: the non-volatile mycotoxin gliotoxin and the mono-functional alkylating agent methyl methanesulfonate (MMS) were studied. Concentration-response curves for each agent were established and the IC50 value (concentration resulting in 50% inhibition of colony growth or absorbance) was estimated. There are differences in toxicity levels between the MVOC tested and gliotoxin and MMS. The most toxic MVOC was 1-decanol which was as effective as MMS in all test systems. 1-decanol was about 10-fold more toxic than the other MVOC. All MVOC tested were more than 1000-fold less toxic than gliotoxin.

Expression of vascular endothelial growth factor and its receptors in multiple myeloma and other hematopoietic malignancies

Vascular endothelial growth factor (VEGF) is a potent angiogenic peptide with biologic effects that include regulation of hematopoietic stem cell development, extracellular matrix remodeling, and inflammatory cytokine generation. The importance of angiogenic factors such as VEGF, while clearly established in solid tumors, has not been fully elucidated in human hematopoietic neoplasms. Human hematopoietic tumor cell lines, representing multiple lineages and diseases, produce and secrete VEGF and express at least one of its two receptors. Exposure of human vascular endothelial cells to VEGF increased the expression of several hematopoietic growth factors known to be involved in myeloma including interleukin-6 (IL-6). Bone marrow samples from patients diagnosed with multiple myeloma were examined for expression of VEGF and its receptors. VEGF protein production was detected in malignant plasma cells from 78% of the myeloma patients studied. While expression of the Flt-I and KDR receptors was not observed in the malignant plasma cells, both were markedly elevated in the normal marrow myeloid and monocytic cells surrounding the tumor. In bone marrow clot sections from normal allogeneic donors, low-intensity cytoplasmic VEGF expression was detected infrequently in isolated myelocytes, macrophages, and megakaryocytes. In vitro colony-forming assays using patient-derived material revealed that antibody neutralization of VEGF resulted in an inhibition of colony growth, whereas the addition of recombinant human VEGF stimulated colony formation. Neutralization of VEGF activity also suppressed the generation of tumor necrosis factor-alpha (TNF-α) and interleukin-I β (IL-I β) from bone marrow mononuclear cells. These data raise the possibility that VEGF may play a role in the growth of hematopoietic neoplasms such as multiple myeloma through paracrine and perhaps autocrine mechanisms.