The influence of reduced glutathione in fertilization medium on the fertility of in vitro-matured C57BL/6 mouse oocytes.

It is well known that IVM oocytes show a decreased potential for fertility and development compared with in vivo-matured oocytes. In this study, we added reduced glutathione (GSH) to the fertilization medium during IVF to investigate its effect on the fertility and early embryo development of IVM oocytes. The fertilization rate for IVM oocytes and fresh sperm increased with the addition of GSH (0, 1.0, and 2.0 mM: 51%, 76%, and 70%). Moreover, the addition of GSH to the fertilization medium also improved the developmental potential compared with the control sample (0 mM). In addition, we performed IVF using IVM oocytes and frozen/thawed sperm that had been cryopreserved in a mouse bank. Results indicated a marked increase in the fertilization rate when 1.0 mM GSH was added to the fertilization medium compared with when no GSM was used (0.0 mM GSH: 2% (3/195); 1.0 mM GSH: 33% (156/468)). Furthermore, the fertilization rate improved dramatically via zona drilling using laser equipment (52%: 267/516), whereas normal offspring were obtainsed after transferring embryos created via IVF using IVM oocytes and frozen/thawed sperm. This is the first report in which offspring have been obtained via IVF using IVM oocytes and frozen/thawed sperm.

Combination medium of cryoprotective agents containing L-glutamine and methyl-{beta}-cyclodextrin in a preincubation medium yields a high fertilization rate for cryopreserved C57BL/6J mouse sperm.

Recently, a vast number of genetically-engineered mice have been created in various laboratories worldwide, all of which need to be effectively archived. The cryopreservation of mouse sperm provides a simple and economical means of storing the mice in mouse resource facilities. The current protocol for sperm cryopreservation using 18% raffinose pentahydrate and 3% skim milk (R18S3) has been adopted in most laboratories. In general, we can attain relatively high fertilization rates for frozen/thawed sperm in many inbred and F1 hybrid strains. However, the sperm of C57BL/6J mice shows an extremely low fertility rate after freezing and thawing (0-20%). In this study, we attempted to improve the low fertility of frozen/thawed C57BL/6J mouse sperm. Our results showed that a combination of R18S3 containing l-glutamine and methyl-beta-cyclodextrin (MBCD) in a preincubation medium dramatically increased the rate of fertilization (69.2 +/- 12.2%). Furthermore, the developmental potencies of two-cell embryos produced by frozen/thawed sperm to live young were normal (fresh: 46.0 +/- 8.2%, frozen/thawed: 51.5 +/- 11.1%). In summary, we conclude that a new method of sperm cryopreservation and in vitro fertilization using modified R18S3 with l-glutamine and MBCD in a preincubation medium yields a high fertilization rate for frozen/thawed C57BL/6J strain sperm. Furthermore, the new method provides a reliable archiving and reproducing system for genetically-engineered mice using sperm cryopreservation.

Disseminated mucocutaneous herpes simplex virus infection in an immunocompetent woman.

Disseminated mucocutaneous herpes simplex virus (HSV) infection in an immunocompetent person is quite rare. A 19-year-old healthy Japanese woman presented with painful, umbilicated vesicles and pustules on her genital region, both nipples and on the forearm 10 days after the last sexual contact with her partner who had cold sore at that time. Tzanck test and biopsy confirmed the diagnosis of disseminated mucocutaneous HSV infection. She did not have any visceral HSV disease. Skin lesions improved after treatment with acyclovir and erythromycin for seven days. We propose that like herpes gladiatorum, HSV dissemination in this case was acquired by close body contact.

Etretinate enhances the susceptibility of human skin squamous cell carcinoma cells to 5-aminolaevulic acid-based photodynamic therapy.

BACKGROUND: Photodynamic therapy (PDT) with 5-aminolaevulinic acid (5-ALA) is a noninvasive and effective treatment for superficial skin cancers. Etretinate, a derivate of vitamin A, with the chemical formula ethyl(2E,4E,6E,8E)-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethyl-2,4,6,8-nona-tetraenoate, has been reported to have antitumour effects and to regulate the proliferation and differentiation of skin cancers. OBJECTIVE: In order to develop more efficient PDT, we investigated whether etretinate enhanced the cytotoxic action of ALA-based PDT against human squamous cell carcinoma cell line, HSC-5. METHOD: The in vitro cytotoxicity was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptotic cells were detected by double-staining with fluorescent annexin V and propidium iodide. Intracellular protoporphyrin IX (PpIX) converted from exogenous ALA was measured by a fluorescence meter. RESULTS: HSC-5 cells pretreated with a nontoxic concentration of etretinate became more susceptible to the cytotoxic action of ALA-based PDT. Etretinate-pretreated cells underwent apoptosis in response to ALA-based PDT. Etretinate pretreatment resulted in enhanced accumulation of ALA-dependent intracellular PpIX. CONCLUSIONS: The results suggest that etretinate enhances the susceptibility of HSC-5 cells to ALA-based PDT via the intracellular increase of ALA-dependent PpIX. Etretinate might be useful for improvement of ALA-based PDT.

Phorbol ester impairs electrical excitation of rat pancreatic beta-cells through PKC-independent activation of KATP channels.

BACKGROUND: Phorbol 12-myristate 13-acetate (PMA) is often used as an activating phorbol ester of protein kinase C (PKC) to investigate the roles of the kinase in cellular functions. Accumulating lines of evidence indicate that in addition to activating PKC, PMA also produces some regulatory effects in a PKC-independent manner. In this study, we investigated the non-PKC effects of PMA on electrical excitability of rat pancreatic beta-cells by using patch-clamp techniques. RESULTS: In current-clamp recording, PMA (80 nM) reversibly inhibited 15 mM glucose-induced action potential spikes superimposed on a slow membrane depolarization and this inhibition can not be prevented by pre-treatment of the cell with a specific PKC inhibitor, bisindolylmaleimide (BIM, 1 microM). In the presence of a subthreshold concentration (5.5 mM) of glucose, PMA hyperpolarized beta-cells in a concentration-dependent manner (0.8-240 nM), even in the presence of BIM. Based on cell-attached single channel recordings, PMA increased ATP-sensitive K+ channel (KATP) activity. Based on inside-out patch-clamp recordings, PMA had little effect on KATP activity if no ATP was in the bath, while PMA restored KATP activity that was suppressed by 10 microM ATP in the bath. In voltage-clamp recording, PMA enhanced tolbutamide-sensitive membrane currents elicited by repetitive ramp pulses from -90 to -50 mV in a concentration-dependent manner, and this potentiation could not be prevented by pre-treatment of cell with BIM. 4alpha-phorbol 12,13-didecanoate (4alpha-PDD), a non-PKC-activating phorbol ester, mimicked the effect of PMA on both current-clamp and voltage-clamp recording configurations. With either 5.5 or 16.6 mM glucose in the extracellular solution, PMA (80 nM) increased insulin secretion from rat islets. However, in islets pretreated with BIM (1 microM), PMA did not increase, but rather reduced insulin secretion. CONCLUSION: In rat pancreatic beta-cells, PMA modulates insulin secretion through a mixed mechanism: increases insulin secretion by activation of PKC, and meanwhile decrease insulin secretion by impairing beta-cell excitability in a PKC-independent manner. The enhancement of KATP activity by reducing sensitivity of KATP to ATP seems to underlie the PMA-induced impairment of beta-cells electrical excitation in response to glucose stimulation.

Protein kinase C-dependent and -independent inhibition of Ca(2+) influx by phorbol ester in rat pancreatic beta-cells.

Phorbol esters were used to investigate the action of protein kinase C (PKC) on insulin secretion from pancreatic beta-cells. Application of 80 nM phorbol 12-myristate 13-acetate (PMA), a PKC-activating phorbol ester, had little effect on glucose (15 mM)-induced insulin secretion from intact rat islets. In islets treated with bisindolylmaleimide (BIM), a PKC inhibitor, PMA significantly reduced the glucose-induced insulin secretion. PMA decreased the level of intracellular Ca(2+) concentration ([Ca(2+)](i)) elevated by the glucose stimulation when tested in isolated rat beta-cells. This inhibitory effect of PMA was not prevented by BIM. PMA inhibited glucose-induced action potentials, and this effect was not prevented by BIM. Further, 4alpha-phorbol 12,13-didecanoate (4alpha-PDD), a non-PKC-activating phorbol ester, produced an effect similar to PMA. In the presence of nifedipine, the glucose stimulation produced only depolarization, and PMA applied on top of glucose repolarized the cell. When applied at the resting state, PMA hyperpolarized beta-cells with an increase in the membrane conductance. Recorded under the voltage-clamp condition, PMA reduced the magnitude of Ca(2+) currents through L-type Ca(2+) channels. BIM prevented the PMA inhibition of the Ca(2+) currents. These results suggest that activation of PKC maintains glucose-stimulated insulin secretion in pancreatic beta-cells, defeating its own inhibition of the Ca(2+) influx through L-type Ca(2+) channels. PKC-independent inhibition of electrical excitability by phorbol esters was also demonstrated.

2-Aminoethoxydiphenyl borate modulates kinetics of intracellular Ca(2+) signals mediated by inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores in single pancreatic acinar cells of mouse.

Regulation of the kinetics of intracellular Ca(2+) signals with a novel, membrane-penetrable, inositol 1,4,5-trisphosphate (InsP(3)) receptor/Ca(2+) channel modulator, 2-amino-ethoxydiphenyl borate (2APB), has been investigated using patch-clamp, whole-cell recording to monitor Ca(2+)-activated Cl(-) currents in single isolated pancreatic acinar cells. 2APB itself fails to evoke a detectable current response but it dramatically changes the kinetics of agonist-induced Ca(2+) release from pulsatile spikes to long-lasting, huge Ca(2+) waves, suggesting that 2APB coordinates local Ca(2+) release to generate global Ca(2+) signals. The regulation by 2APB can be elicited by internal perfusion of InsP(3) in a concentration-dependent manner, indicating that this regulation is not mediated through membrane receptors or G protein signal transduction. The InsP(3) receptor blocker heparin, but not the ryanodine-sensitive receptor blockers ruthenium red or ryanodine, abolishes 2APB-mediated regulation of Ca(2+) release. This results also suggest that 2APB effects are mediated through InsP(3) receptors. 2APB substantially modifies single inward Cl(-) current pulse evoked by the photolytic release of caged InsP(3) but not by caged Ca(2+). These data indicate that 2APB-induced regulation is mediated neither by Ca(2+)-induced Ca(2+) release nor by affecting Cl(-) channel activity directly. We conclude that 2APB regulates the kinetics of intracellular Ca(2+) signals, represented as the change in the Ca(2+) oscillation patterns from brief pulsatile spikes to huge, long-lasting Ca(2+) waves. Moreover, this regulation seems to be mediated through InsP(3)-sensitive Ca(2+) pools. 2APB may act as a novel, useful pharmacological tool to study the genesis of intracellular Ca(2+) signals.

cAMP-independent decrease of ATP-sensitive K+ channel activity by GLP-1 in rat pancreatic beta-cells.

Using the patch-clamp method, we studied the mechanism of depolarization of rat pancreatic beta-cells induced by glucagon-like peptide 1 (7-36) amide (GLP-1). GLP-1 caused depolarization in a concentration-dependent manner (0.2-100 nM). Exendin (9-39) amide, a GLP-1 receptor antagonist, prevented the GLP-1-induced depolarization. GLP-1 reduced tolbutamide-sensitive membrane currents evoked by voltage ramps from -90 to -50 mV, recorded in the perforated whole-cell configuration, suggesting that GLP-1 decreased the activity of the ATP-sensitive K+ channel (KATP). This GLP-1 effect was prevented by exendin (9-39) amide. In cells treated with Rp-cAMPS, an inhibitor of the cAMP-dependent protein kinase (PKA), GLP-1 still caused depolarization and reduced the whole-cell membrane current through KATP. Examined in the cell-attached configuration, 20 nM GLP-1, applied out of the patch, had little effect on KATP activity. In the inside-out configuration, the open time probability and the single-channel conductance of KATP in the absence of ATP inside the membrane were unaffected by the presence of 20 nM GLP-1 in the pipette. In both conditions, application of ATP to the inside of the membrane reduced KATP activity. The half-maximal concentrations (ki) of ATP were 11.6 microM without and 5.6 microM with 20 nM GLP-1 in the pipette (P<0.05). The values of the Hill coefficient (h) were 1.03 without and 1.01 with GLP-1. We conclude that GLP-1 reduces KATP activity by elevating the sensitivity of KATP to ATP, resulting in depolarization of pancreatic beta-cells. This GLP-1 action is independent of the cAMP signalling pathway.

Protein kinase C-dependent inhibition of K+ currents in noradrenaline-induced depolarization of smooth muscle of guinea-pig vas deferens.

Ionic mechanisms and signal transduction underlying noradrenaline (NA)-induced depolarization in single smooth muscle cells of guinea-pig vas deferens were studied. NA caused depolarization followed by action potentials through activation of 1-adrenoceptors. In the presence of nifedipine, no action potential was generated, and the magnitude of the depolarization depended on the concentration of NA (0.1-100 micrometer). NA, through 1-adrenoceptor activation, reduced the magnitude of membrane currents in response to voltage ramp pulses from -90 to -30 mV in a concentration-dependent manner. The reversal potential of the current inhibited by NA changed proportionally to the change in the equilibrium potential of K+, suggesting that NA inhibited K+ channel activity. Treatment of cells with GDPS, an inhibitor of G proteins, or bisindolylmaleimide (BIM), a selective protein kinase C (PKC) inhibitor, prevented the NA inhibition of the currents. Application of 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of PKC, mimicked the effect of NA. It is suggested that in the smooth muscle of guinea-pig vas deferens, activation of 1-adrenoceptors and the subsequent activation of PKC led to inhibition of K+ currents, which is responsible for the depolarization induced by NA.

Efficacy of pertussis vaccines consisted of antigens detoxified with tea-leaf catechins.

The ability of tea-leaf catechins to detoxify agents was examined. Filamentous hemagglutinin (FHA) and pertussis toxin (PT) were detoxified by the catechins at an extraordinarily lower concentration compared with that of formalin. The sera from the mice immunized by the catechin-treated antigens recognized, not only catechin-treated, but also untreated antigens. Furthermore, catechin-treated PT induced the antibody to neutralize PT activity in the sera of the immunized mice. Pertussis vaccines were prepared including antigens detoxified by the treatment of catechins and intraperitoneally injected into mice. Protection against Bordetella pertussis infection was shown in mice immunized with the vaccines prepared by treatment with catechins. These data suggest that catechins are effective toxoiding agents for preparing a pertussis vaccine.

Hormone-induced secretory and nuclear translocation of calmodulin: oscillations of calmodulin concentration with the nucleus as an integrator.

Many important enzyme activities are regulated by Ca2+-dependent interactions with calmodulin (CaM). Some of the most important targets for CaM action are in the nucleus, and Ca2+-dependent CaM translocation into this organelle has been reported. Hormone-evoked cytosolic Ca2+ signals occur physiologically as oscillations, but, so far, oscillations in CaM concentration have not been described. We loaded fluorescent-labeled CaM into pancreatic acinar cells and monitored the fluorescence in various regions by confocal microscopy. Sustained high concentrations of the hormone cholecystokinin or the neurotransmitter acetylcholine evoked a transient movement of cytosolic CaM from the basal nonnuclear area into the secretory granule region and, thereafter, a more substantial and prolonged translocation of CaM into the nucleoplasm. About 50% of the CaM that bound Ca2+ translocated. At a lower hormone concentration, evoking Ca2+ oscillations, regular spikes of increased CaM concentration were seen in the secretory granule region with mirror image spikes of decreased CaM concentration in the basal nonnuclear region. The nucleus was able to integrate the Ca2+ spike-evoked pulses of CaM translocation into a sustained elevation of the nucleoplasmic concentration of this protein.

Acute myeloid leukemia in the elderly:--159 Nagoya case studies--Nagoya Cooperative Study Group for Elderly Leukemia.

To obtain background information on elderly acute myeloid leukemia (AML), unselected data covering 159 patients aged 60 years or over with AML from 14 hospitals in Nagoya, Japan was analyzed retrospectively. Among these patients, 119 had de novo acute AML, 32 had AML which evolved from myelodysplastic syndrome (MDS-AML), and 8 had other types of leukemia. The survey showed that MDS-AML tended to be more prevalent in patients aged 70 years and older and that MDS-AML showed a significantly more severe degree of leukopenia and anemia than de novo AML. MDS-AML also showed a significantly lower complete remission (CR) rate than that of de novo AML [6.9% (2/29) vs 58.3% (67/11), P < 0.01] and significantly shorter survival times than those of de novo AML [median: 3.6 months vs 9.6 months, P < 0.01 (generalized Wilcoxon test; GW]. In de novo AML, the proportion of patients treated with conventional therapy (CT group) decreased significantly, and that of those with attenuated therapy (AT group) increased significantly as age elevated (P < 0.01). The CT group showed a significantly higher CR rate (65.4% vs 41.2%, P < 0.05) and a significantly longer survival period than those of the AT group [median: 11.6 months vs 4.8 months, P < 0.05 (GW)]. Overall survival rates of the older age groups became significantly shorter with aging [P < 0.01 (GW)].

[Inactivation and toxoiding of biologically-active components of Bordetella pertussis by tea catechins].

An ability of tea catechins known as agents for the disinfection to bacteria and viruses were tested on application for toxoiding biologically active components of Bordetella pertussis. The effects on the activities and antigenicity of filamentous hemagglutinin (FHA) and pertussis toxin (PT) were investigated. The activities of FHA and PT were inactivated by catechins at approximately 10(3) times lower dose (0.2 mM) compared with that of formalin. The activity of inactivated FHA was recovered by dialysis against Tris-HCl buffer, pH 8.0, containing glutathione or Tris-HCl buffer, pH 6.0. But the activity of inactivated PT was not recovered. Antigenicity of catechin-treated antigens were investigated by immunization to mice. The sera from mice immunized by catechin-treated FHA or PT were contained antibody against not only catechin-treated but also non-treated FHA or PT. These results suggest that antigenicity of FHA or PT was not destroyed by the treatment with catechin. We prepared pertussis-component vaccines by treatment of several catechins on the condition that FHA or PT activity was not recovered. Higher efficacy were found on the vaccines made by treatment of epicatechin, epicatechin gallate, or epigallocatechin than those by formalin. The vaccine prepared by using epigallocatechin gallate had significant efficacy as well as that by formalin treated one. From these results, it is suggested that tea leaf catechins were effective agents for toxoiding of vaccine components.

Relationship between grade of intraductal component of breast cancer within main tumor and extent of intraductal spread in surrounding tissue.

Ninety-three specimens obtained by quadrantectomy of invasive ductal carcinoma of the breast were examined to evaluate the correlation between the grade of the intraductal component within the tumor and the extent of intraductal spread in the surrounding tissue. We used immunohistochemistry of a-smooth muscle actin to visualize the contour of intraductal components. The grade of the intraductal component within the tumor was classified into 4 groups (td0 to td3) by counting number of intraductal components; the extent of intraductal spread in the surrounding tissue was also divided into 4 groups (sd0 to sd3) according distance from the tumor margin. Fifty-eight (89.2%) of 65 cases with low-grade td were low-grade sd, and 17 (60.7%) cases of high-grade td were high-grade of sd. The grade of the intraductal component correlated with the extent of intraductal spread in the surrounding tissue, significantly (p < 0.001, Spearman rank correlation test). From these results, we believe that investigation of intraductal components within the tumor is useful for estimating the extent of intraductal spread in the surrounding tissue, and excision specimens from diagnostic lumpectomy should be examined for the extent of the intraductal component, the results being important in estimating the risk of local recurrence.

Influence of drinking green tea on breast cancer malignancy among Japanese patients.

Inhibitory effects of green tea on carcinogenesis have been investigated in numerous laboratory studies using (-)-epigallocatechin gallate (EGCG) or crude green tea extract, and there is also some epidemiologic evidence. Further, EGCG has been reported to inhibit the growth of cancer cells, lung metastasis in an animal model, and urokinase activity. In this study, we first examined the association between consumption of green tea prior to clinical cancer onset and various clinical parameters assessed at surgery among 472 patients with stage I, II, and III breast cancer. We found that increased consumption of green tea was closely associated with decreased numbers of axillary lymph node metastases among premenopausal patients with stage I and II breast cancer and with increased expression of progesterone receptor (PgR) and estrogen receptor (ER) among postmenopausal ones. Since these are potential prognostic factors, we then investigated the prognosis of breast cancer with special reference to consumption of green tea, in a follow-up study of these patients. We found that increased consumption of green tea was correlated with decreased recurrence of stage I and II breast cancer (P < 0.05 for crude disease-free survival); the recurrence rate was 16.7 or 24.3% among those consuming > or = 5 cups or < or = 4 cups per day, respectively, in a seven-year follow-up of stage I and II breast cancer, and the relative risk of recurrence was 0.564 (95% confidence interval, 0.350-0.911) after adjustment for other lifestyle factors. However, no improvement in prognosis was observed in stage III breast cancer. Our results indicate that increased consumption of green tea prior to clinical cancer onset is significantly associated with improved prognosis of stage I and II breast cancer, and this association may be related to a modifying effect of green tea on the clinical characteristics of the cancer.

Kinetics of Ca2+ release evoked by photolysis of caged InsP3 in rat submandibular cells.

Quantitative time-resolved measurements of cytosolic Ca2+ release by photolysis of caged InsP3 have been made in single rat submandibular cells using patch clamp whole-cell recording to measure the Ca2+-activated Cl- and K+ currents. Photolytic release of InsP3 from caged InsP3 at 100 Joules caused transient inward (V(H) = 60 mV) and outward (V(H) = 0 mV) currents, which were nearly symmetric in their time course. The inward current was reduced when pipette Cl- concentration was decreased, and the outward current was suppressed by K+ channel blockers, indicating that they were carried by Cl- and K+, respectively. Intracellular pre-loading of the InsP3 receptor antagonist heparin or the Ca2+ chelator EGTA clearly prevented both inward and outward currents, indicating that activation of Ca2+-dependent Cl- and K+ currents underlies the inward and the outward currents. At low flash intensities, InsP3 caused Ca2+ release which normally activated the K+ and Cl- currents in a mono-transient manner. At higher intensities, however, InsP3 induced an additional delayed outward K+ current (I[K,(delay)]). I[K(delay)] was independent of the initial K+ current, independent of extracellular Ca2+, inhibited by TEA, and gradually prolongated by repeated flashes. The photolytic release of Ca2+ from caged Ca2+ did not mimic the I[K(delay)]. It is suggested that Ca2+ releases from the InsP3-sensitive pools in an InsP3 concentration-dependent manner. Low concentrations of InsP3 induce the transient Ca2+-dependent Cl- and K+ currents, which reflects the local Ca2+ release, whereas high concentrations of InsP3 induce a delayed Ca2+-dependent K+ current, which may reflect the Ca2+ wave propagation.

[A case of papillary adenocarcinoma of the stomach with liver metastases and carcinomatous peritonitis treated effectively by methotrexate/5-fluorouracil sequential chemotherapy].

A 54-year-old male was admitted to receive treatment by anticancer drug for advanced gastric cancer with liver metastases and carcinomatous peritonitis. Upper gastrointestinal series showed a Borrmann type 4 gastric cancer occupying the body and antrum, and a pathological diagnosis of papillary adenocarcinoma was made by endoscopic biopsy. Two cycles of neoadjuvant chemotherapy consisting of 5-FU and low-dose CDDP (FP therapy) were given. No effect was observed, so that the next chemotherapy schedule of sequential MTX/5-FU therapy was performed. We adopted the intermediate (MTX: 100 mg/m2 and 5-FU 600 mg/m2 weekly) dose regimen. When six courses of this regimen were completed, the primary lesion of the stomach was decreased to 60% and the metastatic liver tumor to 72%, basel on criteria for the evaluation of clinical effects of solid cancer chemotherapy. However, pylorous stenosis remained, and we performed gastrojejunostomy and biopsy of the regional lymph node to determine the response to the chemotherapy. A pathological examination of lymph node revealed metastatic papillary adenocarcinoma with xanthogranulomatous change, and was judged as an effect showing grade 2. He was discharged and had the outpatient hospital treatment. He died of exacerbation of carcinomatous peritonitis 10 months after his initial admission. The response duration for sequential MTX/5-FU therapy was 4 months. This therapy was usually effective in patients with poorly differentiated adenocarcinoma of the stomach. Our result indicates that this therapy can be effective for not only poorly differentiated adenocarcinoma but also papillary adenocarcinoma of the stomach in an advanced stage.

[CD7+, CD34+, electronmicroscopically peroxidase-negative acute leukemia transformed from polycythemia vera after 12 years follow-up].

We reported a 72-year-old female patient who developed acute leukemia following a long course of polycythemia vera (PV). For 12 years she had been treated with phlebotomy, nimustine, busulfan, hydroxyurea and irradiation on splenomegaly. In November 1995, her peripheral blood smear showed blast of 30%. Bone marrow blasts were microscopically as well as electromicroscopically peroxidase-negative and CD7 and HLA-DR positive. Six months later, the blasts were positive for CD7, CD34 and HLA-DR. On the basis of morphologic, biochemical and immunophenotypic features, the patient was diagnosed acute leukemia, probably arising at a primitive multipotential stem cell level. She failed to respond to the various combination therapy including prednisolone, vincristine, cytarabine, daunorubicin and etoposide. The stem-cell-leukemia transformation in PV occurs rarely and may be refractory to chemotherapy.