Eosinophilic differentiation of the human promyelocytic leukemia cell line, HL-60.

HL-60 promyelocytic leukemia cells differentiated to eosinophils and eosinophilic precursors when cultured under mildly alkaline conditions (pH 7.6-7.8) for 7 d without refeeding. New cytoplasmic granules appeared blue in the least mature cells and red in the most mature cells when stained with Wright-Giemsa. The granules also stained with Luxol-fast-blue, a characteristic of eosinophil granules. Furthermore, most cells contained the eosinophil major basic protein (MBP); the Charcot-Leyden Crystal (CLC) protein (lysophospholipase), eosinophil peroxidase, acid phosphatase, and arylsulfatase were also detected in a portion of these cells. The eosinophil major basic protein was found in a high proportion of undifferentiated cells, and thus may be constituitively produced. By examining finely banded chromosomes, translocation break points were demonstrated at q22 on one chromosome 16 and at q23 on the other homologue; abnormalities in this region of the long arm of 16 are a characteristic finding in the recently described syndrome of acute myelomonocytic leukemia (AMMoL) with abnormal bone marrow eosinophils. In common with the bone marrow eosinophils in these patients, the HL-60 eosinophil granules contained chloroacetate esterase and periodic-acid Schiff (PAS) reactive material; crystalloid inclusions were rare. Therefore, the HL-60 cell line appears to be an in vitro model for eosinophilopoiesis and may be specially suited for the study of the abnormal eosinophils seen in certain malignant conditions.

OCI-5/GPC3, a glypican encoded by a gene that is mutated in the Simpson-Golabi-Behmel overgrowth syndrome, induces apoptosis in a cell line-specific manner.

OCI-5/GPC3 is a member of the glypican family. Glypicans are heparan sulfate proteoglycans that are bound to the cell surface through a glycosyl-phosphatidylinositol anchor. It has recently been shown that the OCI-5/GPC3 gene is mutated in patients with the Simpson-Golabi-Behmel Syndrome (SGBS), an X-linked disorder characterized by pre- and postnatal overgrowth and various visceral and skeletal dysmorphisms. Some of these dysmorphisms could be the result of deficient growth inhibition or apoptosis in certain cell types during development. Here we present evidence indicating that OCI-5/GPC3 induces apoptosis in cell lines derived from mesothelioma (II14) and breast cancer (MCF-7). This induction, however, is cell line specific since it is not observed in NIH 3T3 fibroblasts or HT-29 colorectal tumor cells. We also show that the apoptosis-inducing activity in II14 and MCF-7 cells requires the anchoring of OCI-5/GPC3 to the cell membrane. The glycosaminoglycan chains, on the other hand, are not required. MCF-7 cells can be rescued from OCI-5/GPC3-induced cell death by insulin-like growth factor 2. This factor has been implicated in Beckwith-Wiedemann, an overgrowth syndrome that has many similarities with SGBS. The discovery that OCI-5/GPC3 is able to induce apoptosis in a cell line- specific manner provides an insight into the mechanism that, at least in part, is responsible for the phenotype of SGBS patients.

A retroviral oncogene, akt, encoding a serine-threonine kinase containing an SH2-like region.

The v-akt oncogene codes for a 105-kilodalton fusion phosphoprotein containing Gag sequences at its amino terminus. Sequence analysis of v-akt and biochemical characterization of its product revealed that it codes for a protein kinase C-related serine-threonine kinase whose cellular homolog is expressed in most tissues, with the highest amount found in thymus. Although Akt is a serine-threonine kinase, part of its regulatory region is similar to the Src homology-2 domain, a structural motif characteristic of cytoplasmic tyrosine kinases that functions in protein-protein interactions. This suggests that Akt may form a functional link between tyrosine and serine-threonine phosphorylation pathways.

Re-expression of the tumor suppressor NF2/merlin inhibits invasiveness in mesothelioma cells and negatively regulates FAK.

The neurofibromatosis type 2 NF2 gene product, merlin, is a tumor suppressor frequently inactivated in malignant mesothelioma (MM). To investigate a possible correlation between merlin inactivation and MM invasiveness, we restored merlin expression in NF2-deficient MM cells. Re-expression of merlin markedly inhibited cell motility, spreading and invasiveness, properties connected with the malignant phenotype of MM cells. To test directly whether merlin inactivation promotes invasion in a nonmalignant system, we used small interfering RNA to silence Nf2 in mouse embryonic fibroblasts (MEFs) and found that downregulation of merlin resulted in enhanced cell spreading and invasion. To delineate signaling events connected with this phenotype, we investigated the effect of merlin expression on focal adhesion kinase (FAK), a key component of cellular pathways affecting migration and invasion. Expression of merlin attenuated FAK phosphorylation at the critical phosphorylation site Tyr397 and disrupted the interaction of FAK with its binding partners Src and p85, the regulatory subunit of phosphatidylinositol-3-kinase. In addition, NF2-null MM cells stably overexpressing FAK showed increased invasiveness, which decreased significantly when merlin expression was restored. Collectively, these findings suggest that merlin inactivation is a critical step in MM pathogenesis and is related, at least in part, with upregulation of FAK activity.

Cell cycle withdrawal promotes myogenic induction of Akt, a positive modulator of myocyte survival.

During myogenesis, proliferating myoblasts withdraw from the cell cycle, acquire an apoptosis-resistant phenotype, and differentiate into myotubes. Previous studies indicate that myogenic induction of the cyclin-dependent kinase inhibitor p21 results in an inhibition of apoptotic cell death in addition to its role as a negative cell cycle regulator. Here we demonstrate that the protein encoded by the Akt proto-oncogene is induced in C2C12 cells during myogenic differentiation with a corresponding increase in kinase activity. In differentiating cultures, expression of dominant-negative forms of Akt increase the frequency of cell death whereas expression of wild-type Akt protects against death, indicating that Akt is a positive modulator of myocyte survival. Antisense oligonucleotides against p21 block cell cycle withdrawal, inhibit Akt induction, and enhance cell death in differentiating myocyte cultures. Adenovirus-mediated transfer of wild-type or constitutively active Akt constructs confer partial resistance to cell death under conditions where cell cycle exit is blocked by the antisense oligonucleotides. Collectively, these data indicate that cell cycle withdrawal facilitates the induction of Akt during myogenesis, promoting myocyte survival.

Analysis of the interaction of the novel RNA polymerase II (pol II) subunit hsRPB4 with its partner hsRPB7 and with pol II.

Under conditions of environmental stress, prokaryotes and lower eukaryotes such as the yeast Saccharomyces cerevisiae selectively utilize particular subunits of RNA polymerase II (pol II) to alter transcription to patterns favoring survival. In S. cerevisiae, a complex of two such subunits, RPB4 and RPB7, preferentially associates with pol II during stationary phase; of these two subunits, RPB4 is specifically required for survival under nonoptimal growth conditions. Previously, we have shown that RPB7 possesses an evolutionarily conserved human homolog, hsRPB7, which was capable of partially interacting with RPB4 and the yeast transcriptional apparatus. Using this as a probe in a two-hybrid screen, we have now established that hsRPB4 is also conserved in higher eukaryotes. In contrast to hsRPB7, hsRPB4 has diverged so that it no longer interacts with yeast RPB7, although it partially complements rpb4- phenotypes in yeast. However, hsRPB4 associates strongly and specifically with hsRPB7 when expressed in yeast or in mammalian cells and copurifies with intact pol II. hsRPB4 expression in humans parallels that of hsRPB7, supporting the idea that the two proteins may possess associated functions. Structure-function studies of hsRPB4-hsRPB7 are used to establish the interaction interface between the two proteins. This identification completes the set of human homologs for RNA pol II subunits defined in yeast and should provide the basis for subsequent structural and functional characterization of the pol II holoenzyme.

Assessment of methods for the cytogenetic analysis of human solid tumors.

A comparative study was undertaken to evaluate the effectiveness of various procedures (e.g., direct harvest vs. short-term culture) for the cytogenetic analysis of human solid tumors. A total of 51 specimens (38 primary tumors, 13 effusions) were examined from 45 patients with tumors of the ovary, lung, breast, colon, and testicles. Sufficient numbers (greater than or equal to 3) of metaphase cells to be useful for karyotypic analysis were obtained in 42 of 51 (82.3%) specimens. More than 10 analyzable metaphases were found in each of 32 specimens (62.7%), but in some cases it was necessary to examine many slide preparations to achieve this number. The rate of successful chromosome preparations was markedly better for short-term cultures than for specimens harvested directly. Short-term cultures generally showed a mitotic peak after about 3 days of growth in vitro. Generally, the quality of metaphase cells was better in specimens disaggregated enzymatically with collagenase than in those dissociated mechanically. Exposure to ethidium bromide (EB) for the final 2 hours of culture yielded more cells with elongated chromosomes than cultures harvested by conventional methods without EB. Overall, the findings indicate that successful karyotypic analysis can be performed in a high percentage of human solid tumors with the use of techniques that can be readily applied in most cytogenetics laboratories. However, further methodological advances in tissue culture are warranted to routinely provide large numbers of mitotic cells for karyotypic analysis.

Characterization of MOAT-C and MOAT-D, new members of the MRP/cMOAT subfamily of transporter proteins.

BACKGROUND: Multidrug resistance-associated protein (MRP) and canalicular multispecific organic anion transporter (cMOAT) are transporter proteins that pump organic anions across cellular membranes and have been linked to resistance to cytotoxic drugs. We previously identified MOAT-B, an MRP/cMOAT-related transporter, by use of a polymerase chain reaction approach. However, analysis of expressed sequence tag (EST) databases indicated that there might be additional MRP/cMOAT-related transporters. To further define the MRP/cMOAT subfamily of transporters, we used EST probes to isolate complementary DNAs for two related transporter proteins, MOAT-C and MOAT-D. METHODS: MOAT-C and MOAT-D expression patterns in human tissues were determined by RNA blot analysis, and chromosomal localization of the genes was determined by fluorescence in situ hybridization. RESULTS: MOAT-C is predicted to encode a 1437-amino-acid protein that, among eukaryotic transporters, is most closely related to MRP, cMOAT, and MOAT-B (about 36% identity). However, MOAT-C is less related to MRP and cMOAT than MRP and cMOAT are to each other (about 48% identity). Like MOAT-B, MOAT-C lacks an N-terminal membrane-spanning domain, indicating that the topology of this protein is similarly distinct from that of MRP and cMOAT. MOAT-D is predicted to encode a 1527-amino-acid protein that is the closest known relative of MRP (about 58% identity). MOAT-D is also highly related to cMOAT (about 47% identity). The presence of an N-terminal membrane-spanning domain indicates that the topology of MOAT-D is quite similar to that of MRP and cMOAT. MOAT-C transcripts are widely expressed in human tissues; however, MOAT-D transcript expression is more restricted. The MOAT-C and MOAT-D genes are located at chromosomes 3q27 and 17q21.3, respectively. CONCLUSIONS: On the basis of amino acid identity and protein topology, the MRP/cMOAT transporter subfamily falls into two groups; the first group consists of MRP, cMOAT, and MOAT-D, and the second group consists of MOAT-B and MOAT-C.

Double minute chromosomes in acute myeloblastic leukemia.

Two patients with acute myeloblastic leukemia (AML) with double minute chromosomes (dmins) are described. One patient had dmins in approximately one-third of bone marrow cells examined at diagnosis; no other karyotypic changes were observed. The dmins disappeared when the patient achieved a complete remission. The second patient developed acute leukemia as a second cancer, having previously received radiotherapy and chemotherapy for a breast carcinoma. At the time of diagnosis of AML, the patient exhibited dmins in 12% of bone marrow cells; other complex karyotypic changes were observed. Data on the clinical and cytogenetic features of these cases are compared with those of other reported cases of acute leukemia with dmins. The possible biologic and clinical significance of dmins in acute leukemia is discussed.

Spontaneous transformation of rat ovarian surface epithelial cells: association with cytogenetic changes and implications of repeated ovulation in the etiology of ovarian cancer.

BACKGROUND: Ovarian surface epithelial cells undergo several rounds of division to repair the wound created by follicular rupture at the time of ovulation. This cyclical requirement for cell division, when not interrupted by the long anovulatory rest periods that occur during pregnancy and lactation, may contribute to the development of ovarian cancer. PURPOSE AND METHODS: To test this hypothesis, we isolated rat ovarian surface epithelial cells from 10 adult female Fisher rats, initiated two mixed-population and seven clonal cell lines, and repeatedly subcultured these cells in vitro for more than 20 passages. We then tested them for the acquisition of the following four features associated with transformation: 1) the loss of contact inhibition, 2) the capacity for substrate-independent growth, 3) the ability to form tumors when injected subcutaneously and/or intraperitoneally into athymic mice, and 4) cytogenetic abnormalities. RESULTS: Loss of contact inhibition was observed in all nine late-passage cell lines. Six of the nine late-passage, but none of the early-passage, cell lines tested exhibited a capacity for substrate-independent growth that was augmented in a dose-dependent manner by epidermal growth factor. Two late-passage cell lines (clone 2 and mixed-population 2) generated tumors in athymic BALB/c mice within 3 weeks following subcutaneous injection of 5 x 10(6) cells, whereas similar numbers of early-passage cells from the same cell lines failed to generate palpable tumors. Late-passage clone 7 cells were tumorigenic when 5 x 10(7) cells were injected intraperitoneally. Two of the cell lines analyzed exhibited alterations involving losses of part or all of one member of the chromosome 5 pair. Clone 2 possessed an interstitial deletion, del(5)(q21.3q24), consistent with the loss of an uncloned putative tumor suppressor gene at 5q22q23 previously reported to reside near the loci for the interferon alpha, interferon beta, and c-jun genes. Early-passage clone 7 cells exhibited chromosome 5 monosomy, while late-passage cells contained one normal chromosome 5 and a derivative (5q12q). Southern analysis of the three cell lines revealed no consistent loss of loci for the interferon and c-jun genes, although early-passage clone 7 cells had one half the gene copy number for the interferon beta and c-jun genes and both early- and late-passage clone 7 cells lacked DNA sequences hybridizing with the probe for interferon alpha. CONCLUSION: This pattern of passage-dependent spontaneous transformation of rat ovarian surface epithelial cells in vitro supports the hypothesis that repetitious ovulation contributes to the etiology of human ovarian cancer.

Chromosome abnormalities in human non-small cell lung cancer.

Clonal cytogenetic abnormalities found in 30 non-small cell lung carcinomas (NSCLC), including 28 newly diagnosed primary tumor specimens, are summarized. Multiple chromosome alterations were identified in every case, and 19 of 30 tumors had near-triploid or near-tetraploid karyotypes. Polysomy 7 and partial gains of 7p, including 7p11-p13 (site of the EGFR gene), were particularly frequent, occurring alone or in combination in 26 tumors. Recurrent losses involving 1p, 3p, 6q, 9p, 11p, 15p, and 17p (where the TP53 gene is located) were each seen in 16-25 cases. Five tumors exhibited double minutes, which were associated with amplified MYC1 (1 case) and EGFR (1 case), as determined by Southern analysis. The cytogenetic data were compiled from either short term cultures of tumor tissue harvested within 1-9 days (18 cases) or later harvests performed on long term cultures or cell lines (6 cases); in the other 6 cases results were obtained from both short term and long term cultures. Two studies were performed to validate the use of long term culture for cytogenetic analysis of solid lung tumors. First, in order to determine whether cytogenetic results from cultures are representative of the original tumor, the modal chromosome number of 13 specimens placed into culture was compared to the DNA index of the original tumor tissue, as measured by flow cytometry. The DNA indices of the solid tumor biopsies agreed with the degree of aneuploidy observed by cytogenetic analysis in every case. Second, in 6 cases we performed direct comparisons of karyotypes obtained from cells cultured by both methods. Identical chromosome abnormalities were detected in short term cultures and later harvests of the same specimen. Overall, our findings indicate that tumorigenesis in NSCLC is characterized by the accumulation of multiple chromosome alterations. Furthermore, these data demonstrate that recurrent cytogenetic changes can be identified in NSCLC and that detailed karyotypes from long term cultures are relevant to the original tumor. Chromosome abnormalities detected by these techniques may have clinical and biological significance. However, the complex pattern of karyotypic changes seen in newly diagnosed NSCLC emphasizes the need for future investigations of premalignant bronchial lesions in order to identify primary genetic changes important for early detection and intervention in this aggressive neoplasm.

Multiple regions of allelic loss from chromosome arm 6q in malignant mesothelioma.

Our previous cytogenetic studies of malignant mesotheliomas (MMs) revealed losses from 6q15-21 in approximately 40% of cases, suggestive of recurrent loss of function of a putative tumor suppressor gene(s) located in this chromosome region. To more precisely define the critical region of molecular genetic loss within 6q, we have constructed a high-resolution deletion map of this chromosome arm in 46 MMs. We analyzed 32 microsatellite markers to detect loss of heterozygosity in tumor DNAs. Allelic losses from 6q were observed in a high percentage (61%) of cases. Partial deletions of 6q were identified in 11 cases, and these were used to define four nonoverlapping regions of chromosomal loss: a region involving 6q14-21 (approximately 9 cM; 7 of 11 cases with partial deletions), a region within 6q16.3-21 (approximately 8 cM; 9 cases), a region within 6q21-23.2 (approximately 10 cM; 8 cases), and a distal region located at 6q25 (approximately 13 cM; 9 cases). Most cases exhibited losses from more than one of these regions. We conclude from these data that genomic losses involving 6q in MM are more frequent than previously recognized cytogenetically and that the deletions fall into four discrete locations, suggesting the existence of multiple tumor suppressor loci in 6q that may contribute to the pathogenesis of this malignancy.

Homozygous deletions within 9p21-p22 identify a small critical region of chromosomal loss in human malignant mesotheliomas.

Previous DNA analyses have demonstrated that 9p13-p22 is a frequent site of chromosomal loss in leukemia, glioma, melanoma, and lung and bladder carcinomas. Recent cytogenetic studies have revealed recurrent alterations of 9p in malignant mesothelioma (MM). We have performed gene dosage studies of 23 MM cell lines, using probes for several 9p21-p22 loci (IFNB, IFNA/IFNW, D9S3, D9S126, D9S169, and D9S171), to identify a common region of deletion. Homozygous and/or hemizygous deletions were identified in 19 (83%) cell lines. Homozygous losses (10 cell lines; 43%) occurred most often at the D9S171 and IFNA/IFNW loci. In 8 cell lines, 2 or more of the 9p loci examined were found to be homozygously lost; 2 others displayed homozygous losses only at the D9S171 locus. Results from our deletion mapping analysis suggest that D9S171 is located between IFNA/IFNW and D9S126. The data presented here indicate that allelic loss from 9p21-p22 is a common occurrence in MM and further delineate the location of a putative 9p tumor suppressor gene(s) to a region between IFNA/IFNW and D9S171. These MM cell lines may facilitate efforts to define an even smaller critically deleted region, leading to the eventual cloning and characterization of this gene.

Comparative genomic hybridization analysis detects frequent, often high-level, overrepresentation of DNA sequences at 3q, 5p, 7p, and 8q in human non-small cell lung carcinomas.

Comparative genomic hybridization analysis was used to identify chromosomal imbalances in 20 non-small cell lung carcinoma (NSCLC) biopsies and cell lines. The chromosome arms most often overrepresented were 3q (85%), 5p (70%), 7p (65%), and 8q (65%), which were observed at high copy numbers in many cases. Other common overrepresented sites were 1q, 2p, and 20p. DNA sequence amplification was often observed, with the most frequent site being 3q26 (six cases). Other recurrent sites of amplification included 8q24, 3q13, 3q28-qter, 7q11.2, 8p11-12, 12p12, and 19q13.1-13.2. The most frequent underrepresented segment was 3p21 (50%); other recurrent sites of autosomal loss included 8p21-pter, 15q11.2-13, 5q11.2-15, 9p, 13q12-14, 17p, and 18q21-qter. These regions of copy number decreases are also common sites of allelic loss, further implicating these sites as locations of tumor suppressor genes. Although some of the overrepresented segments harbor known or suspected oncogenes/growth-regulatory genes, we have identified 3q and 5p as new sites that are very frequently overrepresented in NSCLC. These findings could represent entry points for the identification of novel amplified DNA sequences that may contribute to the development or progression of NSCLC.

Cytotoxic and cytodifferentiative components of 6-thioguanine resistance in HL-60 cells containing acquired double minute chromosomes.

As an experimental strategy for potentially dissociating and studying the cytotoxic and cytodifferentiative antileukemic effects of 6-thioguanine (6-TG), cultured human promyelocytic leukemia cells (HL-60) were serially selected for growth in increasing concentrations of 6-TG (0.5 to 50 micrograms/ml). Three acquired characteristics, cytotoxic resistance, cytodifferentiative resistance, and double minute chromosomes (DM), were monitored at successive 6-TG selection levels. Approximately 200-fold resistance to the cytotoxic effect of 6-TG was acquired at the first selection step, and it neither increased at higher 6-TG selection levels nor reverted to greater sensitivity in cells subcultured off of drug. This was due to the irreversible loss of hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity. In contrast, a lesser, not completely quantifiable, degree of resistance developed to the cytodifferentiative effects of the purine nucleobases hypoxanthine and 6-TG which varied as a function of 6-TG selection pressure. Numerous DM, not observed in the parental wild-type HL-60 cells, appeared at 6-TG (0.5 micrograms/ml) selection which varied substantially in parallel with 6-TG selection pressure up to 6-TG (20 micrograms/ml). At higher selection levels (50 micrograms/ml or prolonged culture on 20 micrograms/ml), a marked decrease in DM occurred which was associated with the acquisition of new marker chromosomes. The most consistent marker was a chromosome 6 with additional material in the short arm (6p+); this was noted as a single copy in the basal 6-TG/20 subline but as two copies (trisomy 6; 2p+) in independently selected higher 6-TG-resistant subcultures. These cytogenetic findings suggest the presence of amplified genes which increased in number and shifted from a predominance in extra-chromosomal DM to intrachromosomal sites as a function of 6-TG selection. Among the 6-TG-resistant sublines, there was no change or a decrease in the amplification level of the known amplified oncogene c-myc from that demonstrated in parental HL-60 cells. Although proof requires detailed analyses with specific gene probes, the overall results imply that: (a) the cytotoxic component of the resistance is due to an invariant loss of HPRT which, therefore, is not likely to be related to amplified genes; (b) the cytodifferentiative component of the resistance is due to a positively selectable mechanism which could be directly or indirectly related to 6-TG-selected amplified genes; and (c) variations in the cytogenetic indicators of amplified genes and the resistance to 6-TG cannot be simply ascribed to quantitative variations in c-myc amplification.

Chromosomal DNA cytophotometry in 20q- nonspecific myeloid disorders.

DNA cytophotometry was used to quantify the chromosomal alterations in the bone marrow and blood of three patients with nonspecific myeloid disorders. All patients possessed a population of cells with a morphologically abnormal chromosome 20, del(20)(qll). In two of the patients, the abnormal chromosome 20 showed nearly identical DNA measurements with a net loss of 0.37% of the total autosomal DNA in one patient and 0.38% in the second. The third patient had a net loss of only 0.25% of the autosomal DNA. Analysis of the DNA content of the long arm and short arm of the abnormal No. 20 indicated that all three cases had chromosomal material added to the short arm (0.10 to 0.14% of the autosomal DNA). About 0.50% of the autosomal DNA was deleted from the long arm in two of the patients; only 0.35% of the autosomal DNA was deleted from the long arm in the third case. Within the limit of resolution, there is no evidence that the material lost has been translocated intact to another chromosome. The origin of the 20q- chromosome as the result of an incomplete pericentric inversion is suggested.

Isolation of MOAT-B, a widely expressed multidrug resistance-associated protein/canalicular multispecific organic anion transporter-related transporter.

Multidrug resistance-associated protein (MRP) and canalicular multispecific organic anion transporter (cMOAT) are closely related mammalian ATP-binding cassette transporters that export organic anions from cells. Transfection studies have established that MRP confers resistance to natural product cytotoxic agents, and recent evidence suggests the possibility that cMOAT may contribute to cytotoxic drug resistance as well. Based upon the potential importance of these transporters in clinical drug resistance and their important physiological roles in the export of the amphiphilic products of phase I and phase II metabolism, we sought to identify other MRP-related transporters. Using a degenerate PCR approach, we isolated a cDNA that encodes a novel ATP-binding cassette transporter, which we designated MOAT-B. The MOAT-B gene was mapped using fluorescence in situ hybridization to chromosome band 13q32. Comparison of the MOAT-B predicted protein with other transporters revealed that it is most closely related to MRP, cMOAT, and the yeast organic anion transporter YCF1. Although MOAT-B is closely related to these transporters, it is distinguished by the absence of a approximately 200 amino acid NH2-terminal hydrophobic extension that is present in MRP and cMOAT and which is predicted to encode several transmembrane spanning segments. In addition, the MOAT-B tissue distribution is distinct from MRP and cMOAT. In contrast to MRP, which is widely expressed in tissues, including liver, and cMOAT, the expression of which is largely restricted to liver, the MOAT-B transcript is widely expressed, with particularly high levels in prostate, but is barely detectable in liver. These data indicate that MOAT-B is a ubiquitously expressed transporter that is closely related to MRP and cMOAT and raise the possibility that it may be an organic anion pump relevant to cellular detoxification.

AKT2, a member of the protein kinase B family, is activated by growth factors, v-Ha-ras, and v-src through phosphatidylinositol 3-kinase in human ovarian epithelial cancer cells.

Three members have been identified in the protein kinase B (PKB) family, i.e., Akt/PKB alpha, AKT2/PKB beta, and AKT3/PKB gamma. Previous studies have demonstrated that only AKT2 is predominantly involved in human malignancies and has oncogenic activity. However, the mechanism of transforming activity of AKT2 is still not well understood. Here, we demonstrate the activation of AKT2 with several growth factors, including epidermal growth factor, insulin-like growth factor 1, insulin-like growth factor II, basic fibroblast growth factor, platelet-derived growth factor, and insulin, in human ovarian epithelial cancer cells. The kinase activity and the phosphorylation of AKT2 were induced by the growth factors and blocked by the phosphatidylinositol (PI) 3-kinase inhibitor, wortmannin, and dominant-negative Ras (N17Ras). Moreover, the activated Ras and v-Src, two proteins that transduce growth factor-generated signals, also activated AKT2, and this activation was not significantly enhanced by growth factor stimulation but was abrogated by wortmannin. These results indicate that AKT2 is a downstream target of PI 3-kinase and that Ras and Src function upstream of PI 3-kinase and mediate the activation of AKT2 by growth factors. The findings also provide further evidence that AKT2, in cooperation with Ras and Src, is important in the development of some human malignancies.

Chromosomal alterations in acute leukemia patients studied with improved culture methods.

Cytogenetic studies, using improved short-term culture techniques, were performed on 64 patients with acute leukemia to determine the incidence and kinds of clonal karyotypic changes detectable with this newer methodology. An adequate number of analyzable mitoses was obtained from 59 patients. Clonal chromosomal alterations were found in 88% (52 of 59) of patients, as compared to approximately 50% in previous studies of acute leukemia in which conventional techniques were used. From our series, abnormal karyotypes were detected in 37 of 44 (84%) cases with primary acute nonlymphocytic leukemia, all 5 with secondary acute nonlymphocytic leukemia, and all 10 with acute lymphoblastic leukemia. Among the entire group of patients, several recurrent abnormalities were observed, e.g., -7 in eight cases, +8 in seven cases, t(15;17) in four cases, and t(8;21) or a variant of this translocation in four cases. In five patients, the only abnormality was a rather subtle structural rearrangement (e.g., tiny deletion). Five other patients had clonal changes which were found in less than 10% of the mitoses examined in each case. Our results indicate that most patients with acute leukemia, both acute nonlymphocytic leukemia and acute lymphoblastic leukemia, have clonal chromosome abnormalities associated with their disease.