Complex molecular genetic abnormalities involving three or more genetic mutations are important prognostic factors for acute myeloid leukemia.

We conducted a comprehensive analysis of 28 recurrently mutated genes in acute myeloid leukemia (AML) in 271 patients with de novo AML. Co-mutations were frequently detected in the intermediate cytogenetic risk group, at an average of 2.76 co-mutations per patient. When assessing the prognostic impact of these co-mutations in the intermediate cytogenetic risk group, overall survival (OS) was found to be significantly shorter (P=0.0006) and cumulative incidence of relapse (CIR) significantly higher (P=0.0052) in patients with complex molecular genetic abnormalities (CMGAs) involving three or more mutations. This trend was marked even among patients aged ⩽65 years who were also FLT3-ITD (FMS-like tyrosine kinase 3 internal tandem duplications)-negative (OS: P=0.0010; CIR: P=0.1800). Moreover, the multivariate analysis revealed that CMGA positivity was an independent prognostic factor associated with OS (P=0.0007). In stratification based on FLT3-ITD and CEBPA status and 'simplified analysis of co-mutations' using seven genes that featured frequently in CMGAs, CMGA positivity retained its prognostic value in transplantation-aged patients of the intermediate cytogenetic risk group (OS: P=0.0002. CIR: P<0.0001). In conclusion, CMGAs in AML were found to be strong independent adverse prognostic factors and simplified co-mutation analysis to have clinical usefulness and applicability.

The Bcr-Abl kinase inhibitor INNO-406 induces autophagy and different modes of cell death execution in Bcr-Abl-positive leukemias.

Bcr-Abl tyrosine kinase (TK) inhibitors are promising therapeutic agents for Bcr-Abl-positive (Bcr-Abl(+)) leukemias. Although they are known to promote caspase-mediated apoptosis, it remains unclear whether caspase-independent cell death-inducing mechanisms are also triggered. Here we demonstrated that INNO-406, a second-generation Bcr-Abl TK inhibitor, induces programmed cell death (PCD) in chronic myelogenous leukemia (CML) cell lines through both caspase-mediated and caspase-independent pathways. The latter pathways include caspase-independent apoptosis (CIA) and necrosis-like cell death (CIND), and the cell lines varied regarding which mechanism was elicited upon INNO-406 treatment. We also observed that the propensity toward CIA or CIND in cells was strongly associated with cellular dependency on apoptosome-mediated caspase activity. Cells that undergo CIND have a high apoptosome activity potential whereas cells that undergo CIA tend to have a lower potential. Moreover, we found that INNO-406 promotes autophagy. When autophagy was inhibited with chloroquine or gene knockdown of beclin1 by shRNA, INNO-406-induced cell death was enhanced, which indicates that the autophagic response of the tumor cells is protective. These findings suggest new insights into the biology and therapy of Bcr-Abl(+) leukemias.

Apoptosis-based dual molecular targeting by INNO-406, a second-generation Bcr-Abl inhibitor, and ABT-737, an inhibitor of antiapoptotic Bcl-2 proteins, against Bcr-Abl-positive leukemia.

Bcr-Abl is the cause of Philadelphia-positive (Ph(+)) leukemias and also constitutes their principal therapeutic target, as exemplified by dramatic effects of imatinib mesylate. However, mono-targeting of Bcr-Abl does not always achieve complete leukemia eradication, and additional strategies those enable complete elimination of leukemic cells are desired to develop. Here we demonstrate that INNO-406, a much more active Bcr-Abl tyrosine kinase inhibitor than imatinib, augments the activities of several proapoptotic Bcl-2 homology (BH)3-only proteins (Bim, Bad, Bmf and Bik) and induces apoptosis in Ph(+) leukemia cells via Bcl-2 family-regulated intrinsic apoptosis pathway. ABT-737, an inhibitor of antiapoptotic Bcl-2 and Bcl-X(L), greatly enhanced the apoptosis by INNO-406, even in INNO-406-less sensitive cells with Bcr-Abl point mutations except T315I mutation. In contrast, co-treatment with INNO-406 and other pharmacologic inducers of those BH3-only proteins, such as 17-allylaminogeldanamycin, an heat shock protein-90 inhibitor, or PS-341, a proteasome inhibitor, did not further increase the BH3-only protein levels or sensitize leukemic cells to INNO-406-induced apoptosis, suggesting a limit to how much expression levels of BH3-only proteins can be increased by anticancer agents. Thus, double-barrelled molecular targeting for Bcr-Abl-driven oncogenic signaling and the cell protection by antiapoptotic Bcl-2 family proteins may be the rational therapeutic approach for eradicating Ph(+) leukemic cells.

Her4 mediates ligand-dependent antiproliferative and differentiation responses in human breast cancer cells.

The function of the epidermal growth factor receptor (EGFR) family member HER4 remains unclear because its activating ligand, heregulin, results in either proliferation or differentiation. This variable response may stem from the range of signals generated by HER4 homodimers versus heterodimeric complexes with other EGFR family members. The ratio of homo- and heterodimeric complexes may be influenced both by a cell's EGFR family member expression profile and by the ligand or even ligand isoform used. To define the role of HER4 in mediating antiproliferative and differentiation responses, human breast cancer cell lines were screened for responses to heregulin. Only cells that expressed HER4 exhibited heregulin-dependent antiproliferative responses. In-depth studies of one line, SUM44, demonstrated that the antiproliferative and differentiation responses correlated with HER4 activation and were abolished by stable expression of a kinase-inactive HER4. HB-EGF, a HER4-specific ligand in this EGFR-negative cell line, also induced an antiproliferative response. Moreover, introduction and stable expression of HER4 in HER4-negative SUM102 cells resulted in the acquisition of a heregulin-dependent antiproliferative response, associated with increases in markers of differentiation. The role of HER2 in these heregulin-dependent responses was examined through elimination of cell surface HER2 signaling by stable expression of a single-chain anti-HER2 antibody that sequestered HER2 in the endoplasmic reticulum. In the cell lines with either endogenously (SUM44) or exogenously (SUM102) expressed HER4, elimination of HER2 did not alter HER4-dependent decreases in cell growth. These results suggest that HER4 is both necessary and sufficient to trigger an antiproliferative response in human breast cancer cells.

Membrane associated antitumor effects of crocine-, ginsenoside- and cannabinoid derivates.

In the present work a systematic study was initiated with crocine, ginsenoside and cannabinoid derivatives on multidrug resistant mouse lymphoma cells, viral tumor antigen expression and some human leukocyte functions. Among saffron derivatives, crocin and picrocrocin, triglucosyl and diglucosyl crocetin were ineffective on the reversal of multidrug resistance of lymphoma cells. Ginsenoside increased drug accumulation and tumor antigen expression at 2.0-20.0 micrograms/mL. Some cannabinoid derivatives such as cannabinol, cannabispirol and cannabidiol increased drug accumulation, while cannabidiolic acid, delta-9-THC and tetrahydro-cannabidiolic acid reduced drug accumulation of the human mdr1-gene transfected mouse lymphoma cells. The reversal of multidrug resistance is the result of the inhibition of the efflux pump function in the tumor cells. Crocetin esters were less potent than crocin itself in the inhibition of EBV early antigen expression. However crocin and diglucosylcrocetin inhibited early tumor antigen expression of adenovirus infected cells, but triglucosylcrocetin was less effective at 0.01-1.0 microgram/mL. The crocin had no antiviral effect [on HSV-2 infected vero cells] up to 25 micrograms/mL concentration. Ginsenosides had a moderate inhibitory effect except ginsenoside Rb1 (was the less effective) on the drug efflux pump. Among the cannabinoid derivatives the cannabinol and cannabispirol increased drug accumulation, while cannabidiolic acid and delta-8-THC, delta-9-THC and tetrahydro-cannabinol reduced drug accumulation in multidrug resistant mouse lymphoma cells. It is interesting that ginsenosides had a chemical structure-dependent immunomodulating effect by enhancing the activity of NK-cells and ADCC activities.

Identification of Cannabis pollens using an allergic patient's immunoglobulin E and purification and characterization of allergens in Cannabis pollens.

Cannabis pollen allergens were detected using the serum of an allergic patient. The allergens were then purified by sequential column chromatography (including DE52 cellulose and phenyl-Sepharose CL-4B) and preparative HPLC. The molecular weight of the allergens were determined as 10,050 and 13,706 by matrix-assisted laser desorption/ionization time of flight mass spectrometry. We utilised Western blotting and development of an enzyme-linked immunosorbent assay for the detection of Cannabis pollen allergens.

Effect of benzodiazepine on antigen-induced eosinophil infiltration into guinea pig conjunctiva.

The effect of benzodiazepine (BZP) on experimental allergic conjunctivitis was studied. Male Hartley guinea pigs were sensitized with ovalbumin (OVA) and then homologous anti-OVA serum was injected intravenously into guinea pigs for passive sensitization. BZP was administered at a dose of 5 mg/kg by intraperitoneal injection (i.p.) according to the following schedule respectively; a single, repeated twice a day for 3 days, repeated twice a day for 3 days plus a single and repeated twice a day for 7 days. OVA challenge was performed to the conjunctiva, 20 minutes later, conjunctival edema during the early phase response (EPR) was observed and again at 6 hours later, both eosinophil infiltration into the conjunctiva and the platelet activating factor (PAF) serum level during the late phase response (LPR) were examined. BZP did not inhibit the development of conjunctival edema during the EPR. A single dose of BZP did not inhibit, but repeated doses of BZP for 3 or 7 days significantly suppressed eosinophil infiltration during the LPR. And after repeated doses of BZP for 7 days, all PAF serum levels during the LPR were under the lower detection limit of the assay. These results suggest that BZP has an inhibitory effect on antigen-induced eosinophil infiltration into the conjunctiva during the LPR.

Effects of putative cognitive function-enhancing drugs and dopaminergic agents on somatostatin and neuropeptide Y in rat brain.

Reduced levels of somatostatin and neuropeptide Y (NPY) have been demonstrated in the brain of patients with some organic mental disorders. We designed the present study to test whether drugs thought to be effective in improving cognitive functions in these disorders increased the levels of these two peptides. The drugs were given to normal rats for 14 days to examine chronic effects on regional brain somatostatin and NPY levels. Amantadine and bifemelane increased these peptide levels. Idebenone and indeloxazine had little effect. The effect of dopaminergic agents on peptide levels was also tested in rats. 1-Dihydroxyphenylalanine increased somatostatin levels in whole brain, and hypothalamic NPY levels, and reduced NPY levels in the cerebral cortex, hippocampus and striatum. Sulpiride increased the levels of NPY in the striatum and brainstem. 6-Hydroxydopamine decreased cortical somatostatin levels, and increased striatal NPY levels. These findings indicate that some agents used to improve cognitive function impairment in organic mental disorders may increase somatostatin and NPY content in the brain of rats. Also, it is suggest that these peptides are under different influences of the dopaminergic system in the brain.

Characterization of cDNAs for stylar transmitting tissue-specific proline-rich proteins in tobacco.

The pistil of flowers is a specialized organ which contains the female gametophytes and provides the structures necessary for pollination and fertilization. Pollen deposited on the stigmatic surface of a compatible plant germinates a pollen tube which penetrates the stigmatic papillae and grows intercellularly through the style towards the ovules in the ovary. Pollen tube growth is largely restricted to the transmitting tissue in the style. Therefore the stylar transmitting tissue is extremely important for the migration of the pollen cell towards the ovary. We have isolated two related cDNAs, transmitting tissue-specific (TTS)-1 and TTS-2, derived from two proline-rich protein (PRP)-encoding mRNAs that accumulate specifically in the transmitting tissue of tobacco. The deduced PRP sequences share similarities with proline-rich cell wall glycoproteins found in a variety of plants. TTS-1 and TTS-2 mRNAs are induced in very young floral buds, accumulate most abundantly during the later stages of flower development when style elongation is the most rapid, and remain at relatively high levels at anthesis. These mRNAs become undetectable in maturing green fruits. In situ hybridization shows that TTS-1 and TTS-2 mRNA accumulation is restricted to the transmitting tissue of the style. The possible roles that these transmitting tissue-specific PRPs may play in maintaining the structural integrity of the style or in the function of this organ is discussed.

A flower-specific cDNA encoding a novel thionin in tobacco.

We isolated a flower-specific cDNA, FST (flower-specific thionin), which encodes a novel thionin from tobacco. Thionins are basic and cysteine (Cys)-rich, low molecular weight proteins found in many plants. They are believed to play a role in plant defense against pathogens. The central domain of the FST protein shares homology with three gamma-thionins. Like other thionin precursors, the FST protein has an N-terminal domain characteristic of a signal peptide and an acidic C-terminal domain. FST mRNA accumulates specifically in developing flowers and its level drops as flowers mature. Transcripts are present in petals, stamens and pistil but are not detectable in sepals. In situ hybridization revealed that FST mRNA is most abundant in the epidermal cells along the adaxial surface of petals, and in the surface cell layers of the carpel and anther walls. If the FST protein indeed has a protective role in flowers, this pattern of spatial distribution of FST mRNA would appear to maximize this effect on the two internal reproductive whorls. A possible biological role for FST is discussed.

Nucleotide sequence analysis of the genomes of the MAV-PS1 and P-PAV isolates of barley yellow dwarf virus.

The MAV-PS1 and P-PAV isolates of barley yellow dwarf virus (BYDV) are serologically related, but not identical. Both are transmitted by the aphid Macrosiphum avenae, but P-PAV is also transmitted by Rhopalosiphum padi. To evaluate the basis for these and other differences, overlapping clones from cDNA libraries representing the genome of each isolate were characterized by restriction enzyme digestion and by hybridization, and subsequently sequenced. Each genome has six positive strand open reading frames (ORFs) which are similar to those identified from a BYDV isolate from Australia (Vic-PAV). The greatest diversity between MAV-PS1 and P-PAV sequences was found in ORFs located in the 3' half of the respective genomes, in particular ORFs 5 and 6, suggesting that these regions of the genome may be involved in the properties that differentiate MAV-PS1 and P-PAV. Sequence comparisons between P-PAV and Vic-PAV showed a high degree of identity in that all ORFs showed greater than 90% amino acid similarity, except ORF6 which had only 69% similarity.

Molecular analysis of an aurea photosynthetic mutant (Su/Su) in tobacco: LHCP depletion leads to pleiotropic mutant phenotypes.

Su is a nuclear encoded, semi-dominant aurea mutation in Nicotiana tabacum L. The homozygous plants (Su/Su) are pale yellow and non-photosynthetic while the heterozygous (Su/+) are photosynthetically competent and have a yellow-green phenotype which is distinct from that of green wild-type plants (+/+). We have examined the RNA and protein levels for a number of nuclear and plastid encoded chloroplast proteins under high and low light plant growth conditions. Under high light conditions, the light-harvesting chlorophyll a/b binding proteins (LHCP) were undetectable in the homozygous Su/Su plants, and the large subunit (LSu) and the small subunit (SSu) of ribulose bisphosphate carboxylase (Rubisco) and cytochrome b559 were severely deficient. However, only the nuclear encoded cab and plastid encoded psbE mRNA (encoding LHCP and cytochrome b559 respectively) were reduced significantly. In heterozygous Su/+ plants, the level of LHCP was reduced to 25% of that in wild-type plants while cab and psbE mRNA, LSu, SSu and cytochrome b559 remained at normal levels, suggesting that LCHP is more immediately affected by the Su mutant gene product than the rest of the photosynthetic proteins and mRNA examined. Under low light conditions, the levels of cab and psbE mRNA, LSu, SSu and cytochrome b559 in homozygous Su/Su plants were equivalent to those in wild-type plants except LHCP which remained undetectable. Similarly, the LHCP level in low light grown Su/+ plants still remained at 25% of wild-type level. These results indicate that the decrease in LHCP is independent of light conditions and has not resulted from photooxidation, whereas the depletion of other proteins and mRNA examined under high light growth conditions is a consequence of photooxidative damage to Su/Su plastids. Furthermore, transgenic Su/Su and Su/+ plants with a cauliflower mosaic virus 35S (CaMV 35S)-cab construct constitutively maintained high levels of cab mRNA but displayed the same pattern of diminished LHCP accumulation as their non-transformed counterparts when grown under both high and low light conditions. These results indicate that the Su mutation primarily causes depletion of LHCP. The depletion of LHCP leads to photooxidative damage which results in decreased cab mRNA levels and other pleiotropic lesions in Su/Su plants.

Role of the 3'-poly(A) sequence in translational regulation of mRNAs in Xenopus laevis oocytes.

We investigated the effect of 3'-noncoding and poly(A) sequences on the translation and stability of membrane-bound mRNA (maize zein) and free mRNA (Xenopus beta-globin) by injecting SP6 transcripts into stage 6 Xenopus oocytes. With zein mRNA, the presence or absence of a 3'-noncoding or poly(A) sequence had little effect on mRNA stability over 24 h, and the 3'-noncoding sequence played essentially no role in mRNA translation. With short periods of incubation, e.g. 1-2 h, the presence or absence of a poly(A) tail also had little effect on zein mRNA translation; but after longer periods, translation of the poly(A-) mRNA was significantly reduced. A similar pattern of mRNA translation was observed for poly(A+) and poly(A-) Xenopus beta-globin mRNAs. These differences in translational efficiency correlated with the formation of maximally loaded polysomes (seven-eight ribosomes/mRNA) for the poly(A+) zein mRNA and a failure to form large polysomes with the poly(A-) zein mRNA. These results are consistent with a model in which the 3'-poly(A) sequence of mRNAs facilitates reinitiation of ribosomes during protein synthesis.

Aggregation of lysine-containing zeins into protein bodies in Xenopus oocytes.

Zeins, the storage proteins of maize, are totally lacking in the essential amino acids lysine and tryptophan. Lysine codons and lysine- and tryptophan-encoding oligonucleotides were introduced at several positions into a 19-kilodalton zein complementary DNA by oligonucleotide-mediated mutagenesis. A 450-base pair open reading frame from a simian virus 40 (SV40) coat protein was also engineered into the zein coding region. Messenger RNAs for the modified zeins were synthesized in vitro with an SP6 RNA polymerase system and injected into Xenopus laevis oocytes. The modifications did not affect the translation, signal peptide cleavage, or stability of the zeins. The ability of the modified zeins to assemble into structures similar to maize protein bodies was assayed by two criteria: assembly into membrane-bound vesicles resistant to exogenously added protease, and ability to self-aggregate into dense structures. All of the modified zeins were membrane-bound; only the one containing a 17-kilodalton SV40 protein fragment was unable to aggregate. These findings suggest that it may be possible to create high-lysine corn by genetic engineering.

Characterization of Polypeptides Corresponding to Clones of Maize Zein mRNAS.

Zeins, the storage proteins of maize (Zea mays) are a complex group of polypeptides encoded by a large multigene family. The alpha-zein proteins, which account for about 70% of the total, show both size and charge heterogeneity. Although clones corresponding to several different alpha zeins have been characterized, it has not been possible to correlate these sequences with individual zein polypeptides. By translating in Xenopus oocytes RNAs transcribed in vitro from cloned zein mRNAs, we were able to identify the encoded proteins among native zeins or zeins synthesized in oocytes with total zein mRNA. There was no correlation between the isoelectric points of these proteins and the homology of their coding DNA sequences, as the proteins encoded by two closely homologous cDNAs migrated with greater charge heterogeneity than those encoded by less homologous clones. In addition, the size of the proteins as determined by SDS polyacrylamide gel electrophoresis did not always correlate with the length of the protein deduced from the DNA sequence. The ability to match cloned zein sequences to individual native proteins will enable the genetic mapping of cloned genes as well as the analysis of their translational regulation.

Synthetic oligonucleotide tails inhibit in vitro and in vivo translation of SP6 transcripts of maize zein cDNA clones.

A maize zein cDNA clone was used to synthesize mRNA with the SP6 in vitro transcription system. Although we obtained full-length transcripts of the cDNA sequence, these were inefficient templates for protein synthesis. Removal of the 5' oligo(G) sequence that was synthesized during the cDNA cloning procedure allowed efficient translation of the mRNAs in a wheat germ cell-free protein synthesis system or in Xenopus laevis oocytes. The alteration in translational efficiency did not result from an interaction of the 5' oligo(G) homopolymer tail with the 3' oligo(C) sequence, as transcripts with or without the oligo(C) tail were translated similarly in both protein synthesis systems. Ribosome interaction with the mRNA may be affected due either to the secondary structure of the oligo(G) sequence itself, or an unusual secondary structure between the oligo(G) sequence and another region in the mRNA. Synthetic oligonucleotides at the 5' end of cloned cDNA sequences may generally be inhibitory for translation of mRNAs transcribed in vitro.