Comparison of filgrastim, pegfilgrastim, and lipegfilgrastim added to chemotherapy for mobilization of CD34+ cells in non-Hodgkin lymphoma patients.

BACKGROUND: Data are limited on the long-acting granulocyte-colony stimulating factors (G-CSFs) pegfilgrastim (PEG) and lipegfilgrastim (LIPEG) compared with filgrastim (FIL) regarding the mobilization efficiency of CD34+ cells, graft cellular composition, and engraftment. STUDY DESIGN AND METHODS: In this prospective nonrandomized study, 36 patients with non-Hodgkin lymphoma received FIL, 67 received PEG, and 16 patients received LIPEG as a cytokine after chemotherapy. We analyzed the mobilization and collection of CD34+ cells, cellular composition of blood grafts, and hematologic recovery after auto-SCT according to the type of G-CSF used. RESULTS: Patients in the LIPEG group had fewer apheresis sessions (1 vs. 2, p = 0.021 for FIL and p = 0.111 for PEG) as well as higher median blood CD34+ cell counts at the start of the first apheresis (LIPEG 74 × 106 /L vs. FIL 31 × 106 /L, p = 0.084 or PEG 27 × 106 /L, p = 0.021) and CD34+ yields of the first apheresis (FIL 5.1 × 106 /kg vs. FIL 2.3 × 106 /kg, p = 0.105 or PEG 1.8 × 106 /kg, p = 0.012). Also, the costs associated with G-CSF mobilization and apheresis were lower in the LIPEG group. The graft composition was comparable except for the higher infused CD34+ cell counts in the LIPEG group. The engraftment kinetics were significantly slower in the FIL group. CONCLUSION: LIPEG appears to be more efficient compared with PEG after chemotherapy to mobilize CD34+ cells for auto-SCT demonstrated as fewer sessions of aphereses needed as well as 2.8-fold CD34+ cell yields on the first apheresis day. Early hematologic recovery was more rapid in the LIPEG group. Thus further studies on LIPEG in the mobilization setting are warranted.

Preemptive plerixafor injection added to pegfilgrastim after chemotherapy in non-Hodgkin lymphoma patients mobilizing poorly.

Filgrastim is usually combined with chemotherapy to mobilize hematopoietic progenitor cells in non-Hodgkin lymphoma (NHL) patients. Limited information is available on the efficacy of a preemptive plerixafor (PLER) injection in poor mobilizers after chemotherapy and pegfilgrastim. In this prospective study, 72 patients with NHL received chemotherapy plus pegfilgrastim, and 25 hard-to-mobilize patients received also PLER. The usefulness and efficacy of our previously developed algorithm for PLER use in pegfilgrastim-containing mobilization regimen were evaluated as well as the graft cellular composition, hematological recovery, and outcome after autologous stem cell transplantation (auto-SCT) according to the PLER use. A median 3.4-fold increase in blood CD34+ cell counts was achieved after the first PLER dose. The minimum collection target was achieved in the first mobilization attempt in 66/72 patients (92%) and 68 patients (94%) proceeded to auto-SCT. An algorithm for PLER use was fulfilled in 76% of the poor mobilizers. Absolute numbers of T-lymphocytes and NK cells were significantly higher in the PLER group, whereas the number of CD34+ cells collected was significantly lower. Early neutrophil engraftment was slower in the PLER group, otherwise hematological recovery was comparable within 12 months from auto-SCT. No difference was observed in survival according to the PLER use. Chemotherapy plus pegfilgrastim combined with preemptive PLER injection is an effective and convenient approach to minimize collection failures in NHL patients intended for auto-SCT. A significant effect of PLER on the graft cellular composition was observed, but no difference in outcome after auto-SCT was detected.

Kinetics of blood CD34(+) cells after chemotherapy plus G-CSF in poor mobilizers: implications for pre-emptive plerixafor use.

Mobilization and collection of stem cells is difficult in a proportion of patients intended for autologous stem cell transplantation (ASCT). We have evaluated mobilization kinetics of blood CD34(+) cells (B-CD34(+)) to form basis for algorithm to facilitate rational pre-emptive plerixafor use. Altogether 390 chemomobilized patients were included.Forty-three patients (11%) did not reach BCD34+count ≥10×10(6)/l. Mobilization kinetics differed according to the mobilization capacity observed. Among those who were very poor or inadequate mobilizers (peak BCD34(+)count ≤5×10(6)/l and 6­10×10(6)/l, respectively), BCD34+counts rarely rose after white blood cells (WBC) >5­10×10(9)/l, whereas in many standard mobilizers a later rise in CD34(+) counts could be observed. Four algorithms based on WBC and CD34(+) counts were constructed. According to this patient series, algorithm II (WBC >5×109/l and BCD34+≤10×10(6)/l) and algorithm IV (WBC >10×10(9)/l andB-CD34(+) ≤10×10(9)/l) were the most applicable. For algorithm II the sensitivity was 0.97 and specificity 1.00, respectively, to identify patients for plerixafor use provided that all patients with B-CD34+ maximum ≤10×10(6)/l would have needed plerixafor.This simple model needs a prospective validation.

Pre-emptive plerixafor injection increases blood neutrophil, lymphocyte and monocyte counts in addition to CD34+ counts in patients with non-Hodgkin lymphoma mobilizing poorly with chemotherapy plus G-CSF: potential implications for apheresis and graft composition.

BACKGROUND: CXCR4 receptor antagonist plerixafor is used to mobilize hematopoietic stem cells. No detailed data regarding the effects of plerixafor on other blood cell components have been published but may be of importance in regard to graft composition collected after plerixafor injection. PATIENTS AND METHODS: The study included thirty-nine patients with non-Hodgkin lymphoma (NHL) mobilized with chemotherapy plus G-CSF. Plerixafor was given pre-emptively in twenty patients due to poor mobilization or low collection yield. Nineteen NHL patients served as controls. We evaluated CD34(+) counts and WBC counts and differential from the morning of the first plerixafor injection and 8h after the plerixafor injection. From the control patients the corresponding values were evaluated on the morning of the first apheresis and 24h before it. RESULTS: The first plerixafor dose increased CD34(+) counts and number of leukocytes, neutrophils, lymphocytes, eosinophils and monocytes. Leukocyte, neutrophil, lymphocyte, monocyte and eosinophil counts were higher after plerixafor injection compared to the control group at the time of the first apheresis. Minimal graft (⩾2×10(6)/kg CD34(+) cells) was collected in 85% of plerixafor treated patients, with a single apheresis in 45% of the patients. DISCUSSION: Plerixafor significantly increases B-CD34(+) cell counts on the next morning making effective blood stem cell collection possible in the majority of the patients mobilizing poorly. It also influences other blood cell components but impact of this observation in regard to graft content and post-transplant course needs to be assessed in further studies.

Activation and relocalization of caspase 3 during the apoptotic cascade of human mesothelioma cells.

Apoptosis plays an important role in cancer biology. We investigated the expression of caspases 3 and 8 in malignant mesothelioma and malignant mesothelioma cell lines and putative changes in their ultrastructural expression prior and after exposure to epirubicin. Further studies were conducted to compare these changes to the localization and expression of the bcl-2 group of proteins bcl-X, bax and mcl-1, and Fas-Fas ligand in the same cells. In the histological samples, caspase 3 and 8 immunoreactivity was seen in 27/37 (73%) and 16/37 (43%) of the mesotheliomas. The immunostaining was cytoplasmic diffuse, granular, and occasionally nuclear. All six mesothelioma cell lines expressed caspases 3 and 8 by immunoblotting. After exposure to epirubicin the extent of apoptosis was increased in all cell lines investigated, being weakest in the most resistant M38K cell line. Immunoelectron microscopy revealed immunogold labeling for caspases 3 and 8 in the mitochondria with the accumulation of caspase 3 in the apoptotic bodies, while the mitochondrial localization of the bcl-2 proteins appeared to be very stable. Fas receptor could be detected by flow cytometry, whereas the most resistant cell line (M38K) lacked Fas ligand when assessed by RT-PCR. These results suggest the importance of caspase 3 during the apoptotic process of mesothelioma cells and indicate that epirubicin-induced apoptosis is independent of the mitochondrial pathway.

Regulation of the acute myeloid leukemia cell line OCI/AML-2 by endothelial nitric oxide synthase under the control of a vascular endothelial growth factor signaling system.

It is generally accepted that the vascular endothelial growth factor (VEGF) signal system has no role in the maintenance of normal blood cell formation, although it obviously regulates the development of primitive hematopoiesis during an early stage of embryogenesis. The VEGF signaling pathway, however, might have some role in malignant hematopoiesis, since malignant hematopoietic cells, including acute myeloid leukemia (AML) cells, have been shown to express VEGF and its receptors. In endothelial cells, the VEGF/Flk-1/KDR signal system is a very important generator of nitric oxide (NO) through the activation of its downstream effectors phosphatidylinositol-3-OH-kinase (PI3-K), Akt kinase and endothelial NO synthase (eNOS). It is known that NO regulates hematopoiesis and modulates AML cell growth. The role of the VEGF signaling pathway in the control of AML cell growth through eNOS, however, has not been studied. By using the OCI/AML-2 cell line, which expresses VEGF receptor-2, ie Flk-1/KDR, eNOS and VEGF, as analyzed by flow cytometry, and produces VEGF into growth medium, as analyzed by ELISA, we showed that the Akt kinase and NOS activities in these cells were decreased by the inhibitors of VEGF, Flk-1/KDR and PI3-K, and NOS activity also by the direct inhibitor of NOS. The decreased NOS activity led to inhibition of clonogenic cell growth and, to some extent, induction of apoptosis. We also found that blast cells of bone marrow samples randomly taken from 14 AML patients uniformly expressed Flk-1/KDR and to varying degrees eNOS and VEGF, as analyzed by immunohistochemistry. We conclude that autocrine VEGF through Flk-1/KDR, by activating eNOS to produce NO through PI3-K/Akt kinase, maintains clonogenic cell growth in the OCI/AML-2 cell line. Since the patient samples did not express VEGF in all cases, it is possible that in vivo the regulatory connection between these two signal systems is also mediated via endocrine VEGF in addition to autocrine or paracrine VEGF.

Mechanisms of mechanical load-induced atrial natriuretic peptide secretion: role of endothelin, nitric oxide, and angiotensin II.

There are three members in the natriuretic peptide hormone family, atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP, brain natriuretic peptide), and C-type natriuretic peptide (CNP), that are involved in the regulation of blood pressure and fluid homeostasis. CNP is found principally in the central nervous system and vascular endothelial cells while ANP and BNP are cardiac hormones. ANP is synthesized mainly in the atria of the normal adult heart, while BNP is produced by both the atria and ventricles. The mechanisms controlling ANP release have been the subject of intense research, and are now fairly well understood. The major determinant of ANP secretion is myocyte stretch. Although much less is known about the factors regulating BNP release from the heart, myocyte stretch has also been reported to stimulate BNP release from both atria and ventricles. However, whether wall stretch acts directly or via factors such as endothelin- , nitric oxide, or angiotensin II liberated in response to distension has not been established. Recent studies show that by stimulating endothelin type A receptors endothelin plays an important physiological role as a mediator of acute-volume load-induced ANP secretion from atrial myocytes in conscious animals. In fact, endogenous paracrine/autocrine factors liberated in response to atrial wall stretch rather than direct stretch appears to be responsible for activation of ANP secretion in response to volume load, as evidenced by almost complete blockade of ANP secretion during combined inhibition of endothelin type A/B and angiotensin II receptors. Furthermore, under certain experimental conditions angiotensin II and nitric oxide may also exert a significant modulatory effect on stretch-activated ANP secretion. The molecular mechanisms by which endothelin-1, angiotensin II, and nitric oxide synergistically regulate stretch-activated ANP release are yet unclear.

Endothelin stimulates basal and stretch-induced atrial natriuretic peptide secretion from the perfused rat heart.

To examine the role of intracellular signals in the regulation of atrial natriuretic peptide (ANP) release, the effects of endothelin (ET), a putative endogenous agonist for voltage-dependent Ca2+ channels on basal and atrial stretch-stimulated ANP release as well as on hemodynamic parameters (perfusion pressure, heart rate, contractile force) in isolated perfused rat hearts were studied. Infusion of ET (0.9 x 10(-9)-2.3 x 10(-9) M) alone for 30 min caused a dose-dependent sustained increase in the perfusate immunoreactive ANP (IR-ANP) concentration and coronary vasoconstriction. An initial inotropic response with a later decrease in the contractile force in response to ET was observed, while heart rate remained unchanged. A phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), known to stimulate protein kinase-C activity, at a dose of 4.6 x 10(-8) M caused a gradual, slowly progressive increase in perfusate IR-ANP levels and a more rapid increase in perfusion pressure. ET, when infused in combination with TPA, enhanced IR-ANP secretion induced by the phorbol ester. When hearts from spontaneously hypertensive rats (SHR) were examined, the vasoconstrictor response to infusion of ET was greater than that in the normotensive Wistar-Kyoto (WKY) rats. Infusion of eguipressor doses of ET increased the release of IR-ANP in WKY rats, but had no effect on perfusate IR-ANP levels in SHR. To examine effects of ET on stretch-stimulated ANP release, the modified perfused rat heart preparation that enabled the stepwise distension of the right atrium was used. The increase in right atrial pressure (2.65 +/- 0.13 mm Hg) was accompanied by an increase in the perfusate IR-ANP concentration (from 8.3 +/- 1.1 to 13.9 +/- 2.0 ng/5 min; P less than 0.05; n = 15). The increase in right atrial pressure during the ET infusions resulted in a significantly greater increase in the perfusate IR-ANP concentration than vehicle alone. The calculated ANP increase corresponding to the 2-mm Hg increase in the right atrial pressure was 1.52-fold in the control group and 1.74-fold when 1.9 x 10(-9) M ET was infused (P less than 0.05). This study shows that ET stimulates both basal and atrial stretch-stimulated ANP secretion from the isolated perfused heart and suggests that ET is involved in the regulation of stretch-induced ANP release. The results further confirm the potent vasoconstrictor and cardiac effects of ET.

Atrial stretch induces rapid increase in brain natriuretic peptide but not in atrial natriuretic peptide gene expression in vitro.

Pressure and volume overload in vivo is characterized by induction of the expression of two cardiac hormones, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), but whether stretch directly or other pathophysiological factors associated with cardiac overload cause the activation of these genes is not known. In the present study we examined the effect of short-term (from 30 min to 2 h) direct myocardial stretch on atrial ANP and BNP synthesis and release in modified perfused rat heart preparation that enabled the stepwise distension of the right atrium by pressures approximating those found in vivo. The increase in right atrial pressure by 3.6 mm Hg for 2 h resulted in a 3.3- (p < 0.001) and 1.7-fold (p < 0.02) increase in the rate of IR-ANP and IR-BNP release, respectively, into the perfusate. The maximal increase in both ANP and BNP release was seen after 20 min distension. Thereafter the perfusate IR-ANP and IR-BNP concentration gradually decreased, reaching control values within 2 hours. Chromatographic analysis showed that the hearts primarily release the active, processed 28- and 45-amino acid ANP- and BNP-like peptides, respectively, both before and during atrial stretch. Atrial stretch induced rapid stimulation of BNP gene expression: 1.9- (p < 0.001) and 4.5-fold (p < 0.001) increase in right auricular BNP mRNA levels after 1.0 and 2.0 hours' stretching, respectively, was found on Northern blot analysis, while no change was seen after 30 min distension. In contrast, stretching for up to 2 h did not change auricular ANP mRNA, IR-ANP or IR-BNP levels. Our results show for the first time that atrial stretch induces rapid stimulation of both synthesis and secretion of BNP. The induction of BNP gene expression in the very early stages of cardiac overload mimics the induction of protooncogenes and occurred without involvement of humoral or neural factors. The lack of response of atrial ANP mRNA levels demonstrates that the regulation of BNP gene expression differs from that of ANP.

Brain natriuretic peptide in plasma, atria, and ventricles of vasopressin- and phenylephrine-infused conscious rats.

To evaluate the mechanisms of brain natriuretic peptide (BNP) gene expression, we determined the effect of acute cardiac overload (from 30 min to 4 h) on atrial and ventricular BNP mRNA levels in normal and hypertrophied myocardium. Arginine8 vasopressin (AVP; 0.05 microgram/kg.min) and l-phenylephrine (PHE; 20 micrograms/kg.min) were infused iv to increase cardiac workload in conscious spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. At the age of 10-22 months, during the established phase of ventricular hypertrophy, baseline BNP synthesis was increased in the hypertrophic ventricular cells of SHR, as reflected by about 2-fold (P < 0.05-0.001) elevation of levels of immunoreactive BNP (IR-BNP) and BNP mRNA. Intravenous infusions of AVP and PHE increased mean arterial pressure, plasma IR-BNP levels, and ventricular BNP mRNA levels within 1 h of pressure overload; peak levels of BNP mRNA were reached at 4 h. The increase in BNP mRNA levels was slightly greater in the epicardial (2.0- to 2.6-fold; P < 0.01) than in the endocardial layer (1.9- to 2.0-fold; P < 0.01) of the left ventricle. The rapid stimulation of ventricular BNP mRNA synthesis induced by AVP and PHE was accompanied by the simultaneous activation of left atrial BNP gene expression. Left atrial BNP mRNA levels were increased significantly in response to 1-h infusions, and values peaked in both the AVP- and PHE-infused SHR at 2 h, i.e. a 3.6-fold increase in BNP mRNA levels in left atria in AVP-infused SHR, and a 2.5-fold increase in PHE-infused SHR. Right atrial BNP mRNA levels remained unchanged during drug infusion, except for a transient increase in the WKY after 30 min of infusion. The induction of BNP synthesis was also reflected by increased ventricular IR-BNP levels, whereas AVP and PHE did not affect atrial IR-BNP concentrations or contents. In conclusion, the present study shows that pressure overload rapidly stimulates BNP gene expression in the hearts of normal and hypertensive rats. Thus, locally generated BNP in the heart muscle may play a significant role in cardiac adaptation to acute changes in mechanical load.

p53 and redox state in etoposide-induced acute myeloblastic leukemia cell death.

We investigated whether p53, being a redox-sensitive protein, has a role in the responsiveness of AML cells to etoposide. Two subclones of the OCI/AML-2 cell line, the etoposide-sensitive (ES) and the etoposide-resistant (ER), were used as models. Sensitivity to etoposide was measured by trypan blue and annexin V assays. Etoposide-induced peroxide formation was associated with the induction of cell death. Evident expression of mutated p53 was observed in both subclones in basal growth conditions as analysed by Western blotting and flow cytometry. After etoposide exposure for up to 24 hours, some nuclear accumulation of p53 was observed in the ER subclone, as analysed by Western blotting. The conformation of p53, however, was not changed from mutated toward wild-type during exposure in either of the subclones as analysed by flow cytometry. In conclusion, etoposide-induced change in cellular redox state was associated with apoptosis, but was not a sufficient stimulus for p53 to make its conformation active. Thus, mutated p53 seems to have no role in etoposide-induced apoptosis.

Etoposide-induced apoptosis is not associated with the fas pathway in acute myeloblastic leukemia cells.

Two subclones of the OCI/AML-2 cell line, etoposide-sensitive (ES) and etoposide-resistant (ER), established by the authors, were used as models. We investigated whether the Fas pathway is involved in etoposide-induced apoptosis in acute myeloblastic leukemia (AML). Both of the studied subclones expressed the Fas receptor (FasR), but only the ER cell line expressed the Fas ligand (FasL). Etoposide caused an increase in the mean fluorescence intensity of FasR in both subclones, and an induction of FasL in the ES subclone. However, no change in the numbers of apoptotic cells induced by etoposide was observed when FasR was blocked by an antagonist anti-Fas antibody, nor was an agonist anti-Fas antibody alone cytotoxic to the subclones or enhanced the cytotoxic effect of etoposide. The Fas-resistant phenotype of the AML cells was converted to a Fas-sensitive one by cycloheximide (CHX) suggesting the presence of an inhibitory protein of the Fas pathway in the cells. In etoposide-induced apoptosis, the effect of CHX was different, apoptosis-preventing. In conclusion, etoposide-induced apoptosis is not mediated by the Fas pathway in AML.

Effect of endurance training on atrial natriuretic peptide gene expression in normal and hypertrophied hearts.

We studied the effects of physical endurance training on atrial natriuretic peptide (ANP) gene expression in beagle dogs, Wistar rats, and spontaneously hypertensive rats (SHR). The dogs underwent a gradually increased running training up to 40 km/day on a treadmill for 55 wk while the nontrained sibling control dogs were kept in their cages throughout the study. Endurance training caused a significant 13% (P < 0.05) increase in ventricular hypertrophy but did not change plasma immunoreactive (ir)-ANP levels at rest or ventricular ANP mRNA or irANP levels. When normotensive Wistar rats ran up to 2,200 m/day for 8 wk, no significant change was seen in ventricular hypertrophy or in plasma or ventricular irANP levels at rest compared with nontrained controls. However, endurance training caused a 2.2-fold increase in epicardial ANP mRNA levels (P < 0.05). In the SHR strain, running training up to 900 m/day for 31 wk increased ventricular hypertrophy of trained SHR by 7% (P < 0.01) and caused a concomitant 1.6- to 1.7-fold elevation in ventricular irANP and ANP mRNA levels (P < 0.01-0.001) compared with nontrained SHR. In contrast, changes in atrial ANP mRNA or irANP levels in response to training were small in all three protocols. This study shows that in the normal heart induction of ANP synthesis by endurance training is not associated with ventricular hypertrophy. Moreover, the common stimulus for ventricular ANP synthesis induced by both chronic pressure overload and physical training may be mechanical stretching of cardiac myocytes, because endurance training further stimulated ANP synthesis in hypertrophied ventricles in SHR.

Staurosporine, a protein kinase C inhibitor, inhibits atrial natriuretic peptide secretion induced by sarafotoxin, endothelin and phorbol ester.

We studied the effects of two peptides of the endothelin/sarafotoxin family, sarafotoxin-b (SRTX-b) and endothelin (ET-1), as well as the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on immunoreactive atrial natriuretic peptide (IR-ANP) release and on haemodynamic parameters (perfusion pressure, heart rate and contractile force) in isolated perfused rat hearts in order to examine the role of intracellular signals in the regulation of ANP secretion. Infusion of SRTX-b at doses of 0.9 and 2.7 nM for 30 min caused a gradual, dose-dependent increase in IR-ANP release and a more rapid coronary vasoconstriction similar to the infusions of ET-1 (2.7 nM) or TPA (46 nM), known to activate protein kinase C in heart cells. A transient inotropic response with a later decrease in contractile force was observed after infusion of each agent. SRTX-b and TPA produced a sustained chronotropic effect, while ET-1 did not significantly affect the heart rate. Infusion of 100 nM of staurosporine, a potent inhibitor of protein kinase C, did not affect basal IR-ANP release into the perfusion fluid but slightly decreased perfusion pressure, heart rate and contractile force. When infused together with SRTX-b, ET-1 or TPA, staurosporine significantly inhibited the ANP secretion, coronary vasoconstriction and changes in cardiac function induced by the peptides or phorbol ester. This study shows that SRTX-b stimulates ANP release with a potency similar to that of ET-1 and that the kinetics of their effects on ANP secretion resemble those of TPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative analysis of minimal residual disease detection by multiparameter flow cytometry and enhanced ASO RQ-PCR in multiple myeloma.

Multiparameter flow cytometry (MFC) and allele-specific oligonucleotide real-time quantitative PCR (ASO RQ-PCR) are the two most sensitive methods to detect minimal residual disease (MRD) in multiple myeloma (MM). We compared these methods in 129 paired post-therapy samples from 22 unselected, consecutive MM patients in complete/near complete remission. Appropriate immunophenotypic and ASO RQ-PCR-MRD targets could be detected and MRD analyses constructed for all patients. The high PCR coverage could be achieved by gradual widening of the primer sets used for clonality detection. In addition, for 13 (55%) of the patients, reverse orientation of the ASO primer and individual design of the TaqMan probe improved the sensitivity and specificity of ASO RQ-PCR analysis. A significant nonlinear correlation prevailed between MFC-MRD and PCR-MRD when both were positive. Discordance between the methods was found in 32 (35%) paired samples, which were negative by MFC-MRD, but positive by ASO RQ-PCR. The findings suggest that with the described technique, ASO RQ-PCR can be constructed for all patients with MM. ASO RQ-PCR is slightly more sensitive in MRD detection than 6-10-color flow cytometry. Owing to technical demands ASO RQ-PCR could be reserved for patients in immunophenotypic remission, especially in efficacy comparisons between different drugs and treatment modalities.

Cellular signals regulating the release of ANF.

Atrial natriuretic factor (ANF), a peptide hormone that regulates salt and water balance and blood pressure, is synthesized, stored, and secreted from mammalian myocytes. Stretching of atrial myocytes stimulates ANF secretion, but the cellular processes involved in linking mechanical distension to ANF release are unknown. We reported that phorbol esters, which mimic the action of diacylglycerol by acting directly on protein kinase C and the Ca2+ ionophore A23187, which introduces free Ca2+ into the cell, both increase basal ANF secretion in the isolated perfused rat heart. Phorbol ester also increased responsiveness to Ca2+ channel agonists, such as Bay k8644, and to agents that increase cAMP, such as forskolin and membrane-permeable cAMP analogs. In neonatal cultured rat atrial myocytes, protein kinase C activation by 12-O-tetradecanoylphorbol 13-acetate stimulated ANF secretion, whereas the release was unresponsive to changes in intracellular Ca2+. Endothelin, which stimulates phospholipase C mediated hydrolysis of phosphoinositides and activates protein kinase C, increased both basal and atrial stretch-induced ANF secretion from isolated perfused rat hearts. Similarly, phorbol ester enhanced atrial stretch-stimulated ANF secretion, while the increase in intracellular Ca2+ appeared to be negatively coupled to the stretch-induced ANF release. Finally, phorbol ester stimulated ANF release from the severely hypertrophied ventricles of hypertensive animals but not from normal rat myocardium. These results suggest that the protein kinase C activity may play an important role in the regulation of basal ANF secretion both from atria and ventricular cells, and that stretch of atrial myocytes appears to be positively modulated by phorbol esters.

Induction of cardiac natriuretic peptide gene expression in rats trained in hypobaric hypoxic conditions.

Adaptation of cardiac muscle to prolonged hypobaric hypoxia (770-740 mbar, 2,250-2,550 m), endurance training, and their combination was studied in rats by investigating the gene expression of atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) in atria and ventricles. Rats were assigned into the following groups according to the barometric conditions and physical activity; normobaric sedentary (NS), normobaric training, hypobaric sedentary (HS), and hypobaric training (HT). Experimental periods were 10, 21, and 56 days; the groups at 91 days served as recovery groups from exposure to and training in normobaric and hypobaric conditions for 56 days. The right ventricular hypertrophy in HT rats at 10 days and 56 days was associated with elevated BNP mRNA levels (2.1- and 1.7-fold, P < 0.05, respectively), whereas hypobaric exposure without training was not sufficient to significantly increase ventricular BNP gene expression, although it lead to hypertrophy of the right ventricle. Right and left atrial BNP mRNA levels were also increased (up to 3.9-fold, P < 0.01) in 10-day HS and 10-day HT groups. ANP mRNA levels in right ventricle and left ventricular epicardium were over twofold higher (P < 0.05-0.01) in 10-day HS and 10-day HT groups in comparison to 10-day NS group. Plasma immunoreactive ANP concentration was increased (P < 0.05) in both hypobaric groups up to 21 days. The results show that exposure to hypobaric hypoxia itself and endurance training in hypobaric, hypoxic conditions lead to a marked early increase in ventricular and atrial ANP and BNP mRNA levels. The adaptational response to hypoxia was more pronounced when the oxygen availability was lowered additionally by endurance training carried out in hypobaric hypoxic conditions.

An association between mitochondrial function and all-trans retinoic acid-induced apoptosis in acute myeloblastic leukaemia cells.

The present study investigated whether all-trans retinoic acid (ATRA)-induced apoptosis in acute myeloblastic leukaemia (AML) is related to changes in mitochondrial function. Two human AML cell lines, OU-AML-3 and OU-AML-7, known to be inducible to time-dependent apoptosis of varying degrees by ATRA, were used. Apoptosis induced by ATRA was shown to be a slow event. It was detected by the DNA electrophoretic method and cytofluorimetrical annexin V assay after 48 h exposure, and by morphology and polyADPribose polymerase (PARP) cleavage after 72 h exposure of AML cells to ATRA. The efflux of mitochondrial cytochrome c to cytosol was notable in Western blotting after 48 h exposure of the cells to ATRA and was observed before the drop in the mitochondrial membrane potential, which only took place after 72 h exposure, when measured by flow cytometry and a JC-1 probe. The apoptotic events in mitochondria were more evident in the OU-AML-3 than the OU-AML-7 cell line. This might relate to the different bcl-2 contents of the cell lines: the basic bcl-2 levels of the OU-AML-7 cell line were almost twofold compared to that of the OU-AML-3 cell line, as analysed by the ELISA method. However, both of the cell lines showed progressive down-regulation of bcl-2, which began after 12-24 h exposure of the cells to ATRA as determined by ELISA, Western blotting and flow cytometry. The present results show that mitochondria have a role in ATRA-induced apoptosis in AML cells and down-regulation of bcl-2 is related to it. In view of the previously published studies, the present results underline the fact that the timing of apoptotic events, such as fragmentation of DNA, externalization of phosphatidylserine, cytochrome c efflux, change in mitochondrial membrane potential and cleavage of PARP, are, to a notable extent, cell type and inducer-dependent.

p53 pathway in apoptosis induced by all-trans-retinoic acid in acute myeloblastic leukaemia cells.

The role of the p53 pathway in apoptosis induced by all-trans-retinoic acid (ATRA) was studied in 5 human acute myeloid leukaemia (AML) cell lines, OU-AML-3, -4, -5, -7 and -8, previously established and characterized by the authors. Although all the cell lines have a wild-type (wt) p53 gene, the protein is in a mutant conformation detectable by the anti-p53 antibody PAb 240. Exposure of the cell lines to 1.0 microM ATRA for 72 h caused induction of apoptosis detectable by morphology and the annexin V assay. The number of apoptotic cells according to the annexin V assay varied from 16 +/- 8% (OU-AML-7) to 61 +/- 4% (OU-AML-3) in ATRA-treated cells, while it was 7 +/- 6% in control cells. Western blotting and flow cytometry showed down-regulation of the p53 protein by ATRA. The conformation of p53 remained unchanged, being detectable in flow cytometry by PAb 240, but not by PAb 1620 (an antibody which only detects p53 in wt conformation). At the same time bcl-2 was down-regulated as shown by Western blotting and flow cytometry, while no induction of bax was observed by ATRA. On the basis of these results, ATRA-induced apoptosis in these AML cell lines is independent of the p53 pathway, although it is associated with the down-regulation of bcl-2.