Classification of Cryptococcus neoformans and yeast-like fungus isolates from pigeon droppings by colony phenotyping and ITS genotyping and their seasonal variations in Korea.

Cryptococcus neoformans (C neoformans) is a frequent cause of invasive fungal disease in immunocompromised human hosts. Ninety-eight samples of pigeon droppings were collected from the pigeon shelters in Seoul, and cultured on birdseed agar (BSA) and Sabouraud dextrose agar (SDA). One hundred yeast-like colonies were selected and identified via phenotype characteristics, such as colony morphology and biochemical characteristics. This was then followed with genotyping via sequencing of the internal transcribed spacer (ITS) region. The colonies were classified into four kinds of colony color types: brown type (BrT), beige type (BeT), pink type (PT), and white type (WT). Numbers of isolated BrT, BeT, PT, and WT colonies were 22 (22%), 30 (30%), 19 (19%), and 39 (39%), respectively. All BrT colonies were identified as C neoformans. BeT were identified as 19 isolates of Cryptococcus laurentii, 10 isolates of Malassezia furfur, and 1 isolate of Cryptococcus uniguttulatus. PT was divided into two colony color types: light-PT (l-PT) and deep-PT (d-PT). Eighteen of l-PT and one of d-PT were identified as Rhodotorula glutinis and Rhodotorula mucilaginosa, respectively. WT were identified as 34 isolates of Cryptococcus guilliermondii, 3 isolates of Cryptococcus zeylanoides, 1 isolate of Cryptococcus sake, and 1 isolate of Stephanoascus ciferrii. Most strains were classified identically with the use of either phenotype or genotyping techniques, but C uniguttulatus and C sake classified by phenotyping were Pseudozyma aphidis and Cryptococcus famata by genotyping. This rapid screening technique of pathogenic yeast-like fungi by only colony characteristics is also expected to be very useful for primary yeast screening. Additionally, we investigated the seasonal variations of C neoformans and other yeast-like fungi from 379 pigeon-dropping samples that were collected from February 2011 to March 2011. We isolated 685 yeast-like fungi from the samples. Almost all C neoformans and yeast-like fungi were isolated in the fall (298 strains, 43.5%) and spring (244 strains, 35.6%). A few yeast-like fungi were isolated in winter (98 strains, 14.3%) and summer (45 strains, 6%). These results would be used as an important indicator related to epidemiology and prevention of pathogenic yeast-like fungi infections transmitted through pigeon droppings.

Rapid direct identification of Cryptococcus neoformans from pigeon droppings by nested PCR using CNLAC1 gene.

Isolation and identification of Cryptococcus neoformans and pathogenic yeast-like fungi from pigeon droppings has been taken for a long time and requires various nutrients for its growth. In this study, we attempted to establish a rapid direct identification method of Cr. neoformans from pigeon dropping samples by nested-PCR using internal transcribed spacer (ITS) CAP64 and CNLAC1 genes, polysaccharide capsule gene and laccase-associated gene to produce melanin pigment, respectively, which are common genes of yeasts. The ITS and CAP64 genes were amplified in all pathogenic yeasts, but CNLAC1 was amplified only in Cr. neoformans. The ITS gene was useful for yeast genotyping depending on nucleotide sequence. Homology of CAP64 genes among the yeasts were very high. The specificity of PCR using CNLAC1 was demonstrated in Cr. neoformans environmental strains but not in other yeast-like fungi. The CNLAC1 gene was detected in 5 serotypes of Cr. neoformans. The nested-PCR amplified up to 10(-11) µg of the genomic DNA and showed high sensitivity. All pigeon droppings among 31 Cr. neoformans-positive samples were positive and all pigeon droppings among 348 Cr. neoformans-negative samples were negative by the direct nested-PCR. In addition, after primary enrichment of pigeon droppings in Sabouraud dextrose broth, all Cr. neoformans-negative samples were negative by the nested-PCR, which showed high specificity. The nested-PCR showed high sensitivity without culture of pigeon droppings. Nested-PCR using CNLAC1 provides a rapid and reliable molecular diagnostic method to overcome weak points such as long culture time of many conventional methods.

PML suppresses oncogenic transformation of NIH/3T3 cells by activated neu.

The chromosomal translocation t(15;17)(q22;q12) is a consistent feature of acute promyelocytic leukemia (APL) that results in the disruption of genes for the zinc finger transcription factor PML and the retinoic acid receptor alpha (RAR alpha). We have previously shown that PML is a growth suppressor and is able to suppress transformation of NIH/3T3 by activated neu oncogene. In the study presented here, the full-length PML cDNA was transfected into B104-1-1 cells (NIH/3T3 cells transformed by the activated neu oncogene) by retrovirally mediated gene transfer. We found that expression of PML could reverse phenotypes of B104-1-1 including morphology, contact-limiting properties, and growth rate in both transient-expression and stable transfectants. We also demonstrated that PML is able to suppress clonogenicity of B104-1-1 in soft agar assay and tumorigenicity in nude mice. These results strongly support our previous finding that PML is a transformation or growth suppressor. Our results further demonstrate that expression of PML in B104-1-1 cells has little effect on cell cycle distribution. Western blot analysis demonstrated that suppression of neu expression in B104-1-1 by PML was insignificant in the transient transfection experiment but significant in the PML stable transfectants. This study suggests that PML may suppress neu expression and block signaling events associated with activated neu. This study supports our hypothesis that disruption of the normal function of PML, a growth or transformation suppressor, is a critical event in APL leukomogenesis.

Detection of minimal residual cells carrying the t(14;18) by DNA sequence amplification.

By means of the polymerase chain reaction (PCR) technique, DNA sequences were amplified that flank the crossover sites of a characteristic chromosomal translocation for follicular lymphomas, t(14;18)(q32;q21). This technique permitted the detection of cells carrying the t(14;18) hybrid DNA sequences at a dilution of 1:100,000. The remission marrow and blood samples of a patient with follicular lymphoma and the t(14;18) failed to show any abnormality by morphological examination and conventional Southern blot analysis. However, the t(14;18) hybrid DNA sequences were detected by the PCR technique. Thus, this technique is a highly sensitive tool to detect minimal residual cells carrying the t(14;18) and has the potential to identify a subpopulation of patients with subclinical disease.

High-speed characteristics of vertical cavity surface emitting lasers and resonant-cavity-enhanced photodetectors based on intracavity-contacted structure.

We fabricated vertical cavity surface emitting lasers (VCSELs) and resonant-cavity-enhanced photodetectors (RCE-PDs) with GaAs/AlGaAs distributed Bragg reflectors (DBRs), operating at lambda approximately 980 nm, based on an intracavity-contacted structure. The top-DBR mesa diameter of the VCSELs was optimized to 18 microm in terms of slope efficiency, differential series resistance, and 3 dB bandwidth. For VCSELs with an oxide aperture of 4.5 microm and a top-DBR mesa diameter of 18 microm, the threshold current was about 1.2 mA, exhibiting maximum output power of approximately 3.49 mW (at 20 degrees C) with good uniformity. The effect of the overetching in the outermost layer of RCE-PDs on the device performance was also investigated. For RCE-PDs based on the VCSEL structure, a peak responsivity of 0.44 A/W (at lambda approximately 979.7 nm) with a spectral width of approximately 3 nm and a dark current of 68 pA under a bias voltage of -5 V at 20 degrees C was obtained. The maximum 3 dB bandwidths of approximately 11.5 GHz with a modulation current efficiency factor of 5.6 GHz/mA(1/2) at -7 mA and 9 GHz at -7 V were achieved for VCSELs and RCE-PDs, respectively.

Development of microsatellite markers for the Japanese endemic conifer Thuja standishii and transfer to other East Asian species.

OBJECTIVE: Design polymorphic microsatellite loci that will be useful for studies of the genetic diversity, gene-flow and reproduction in the Japanese endemic conifer Thuja standishii and test the transferability of these loci to the two other East Asian species, T. sutchuenensis and T. koraiensis. RESULTS: Fifteen loci were developed which displayed 3 to 21 alleles per locus (average = 9.2) among 97 samples from three populations of T. standishii. Observed heterozygosity for all samples varied between 0.33 and 0.75 (average = 0.54) while expected heterozygosity values were higher with an average over the 15 loci of 0.62 (0.37-0.91). Low multi-locus probability of identity values (< 0.00002) indicate that these markers will be effective for identifying individuals derived from clonal reproduction. All 15 loci amplified in 13 samples of T. sutchuenensis, the sister species of T. standishii, with 1 to 11 alleles per locus (average = 4.33) while 13 loci amplified in four samples of the more distantly related T. koraiensis with 1 to 5 alleles per locus (average = 2.15).

A possibility of detection of the non-charge based analytes using ultra-thin body field-effect transistors.

Ultra-thin body of p-type field-effect transistors were developed as transducer for biosensors. Changes of conductance resulted from the changes of the surface potentials of ultra-thin body field-effect transistors (UTB-FETs) due to surface chemical modifications were demonstrated. The channel surface of UTB-FETs were modified with N-[3-(trimethoxysilyl)propyl]ethylenediamine (AEAPTMS) and then gold nanoparticles (AuNPs) to immobilize the bio-component, the genetically engineered Delta(5)-3-ketosteroid isomerase (Art_KSI) or the Art_KSI conjugated with charged reporter (Art_KSI_mA51). The binding of charge-based molecules or nanoparticles has been demonstrated to strongly affect the conductivity of UTB-FETs; the increase or decrease of the conductance depends on the polarity of the immobilized molecules or nanoparticles. A new protocol involving the detection of a non-charged analyte relied on the competitive binding of analyte (19-norandrostendione) and a charged reporter (mA51) with KSI. When exposed to a 19-norandrostendione solution (10 microM), the conductance of Art_KSI_mA51-modified UTB-FET increased by 265 nS ( approximately 12%). On the other hand, conductance of Art_KSI-modified UTB-FET showed no distinct change under the same detection conditions.

Novel chicken actin gene: third cytoplasmic isoform.

We identified a novel chicken actin gene. The actin protein deduced from its nucleotide sequence very closely resembles the vertebrate cytoplasmic actins; accordingly, we classified this gene as a nonmuscle type. We adopted the convention for indicating the nonmuscle actins of the class Amphibia (Vandekerckhove et al., J. Mol. Biol. 152:413-426) and denoted this gene as type 5. RNA blot analysis demonstrated that the type 5 actin mRNA transcripts accumulate in adult tissues in a pattern indicative of a nonmuscle actin gene. Genomic DNA blots indicated that the type 5 actin is a single copy gene and a distinct member of the chicken actin multigene family. Inspection of the nucleotide sequence revealed many features that distinguished the type 5 gene from all other vertebrate actin genes examined to date. These unique characteristics include: (i) an initiation Met codon preceding an Ala codon, a feature previously known only in plant actins, (ii) a single intron within the 5' untranslated region, with no interruptions in the coding portion of the gene, and (iii) an atypical Goldberg-Hogness box (ATAGAA) preceding the mRNA initiation terminus. These unusual features have interesting implications for actin gene diversification during evolution.

The promyelocytic leukemia protein interacts with Sp1 and inhibits its transactivation of the epidermal growth factor receptor promoter.

The promyelocytic leukemia protein (PML) is a nuclear phosphoprotein with growth- and transformation-suppressing ability. Having previously shown it to be a transcriptional repressor of the epidermal growth factor receptor (EGFR) gene promoter, we have now shown that PML's repression of EGFR transcription is caused by inhibition of EGFR's Sp1-dependent activity. On functional analysis, the repressive effect of PML was mapped to a 150-bp element (the sequences between -150 and -16, relative to the ATG initiation site) of the promoter. Transient transfection assays with Sp1-negative Drosophila melanogaster SL2 cells showed that the transcription of this region was regulated by Sp1 and that the Sp1-dependent activity of the promoter was suppressed by PML in a dose-dependent manner. Coimmunoprecipitation and mammalian two-hybrid assays demonstrated that PML and Sp1 were associated in vivo. In vitro binding by means of the glutathione S-transferase (GST) pull-down assay, using the full-length and truncated GST-Sp1 proteins and in vitro-translated PML, showed that PML and Sp1 directly interacted and that the C-terminal (DNA-binding) region of Sp1 and the coiled-coil (dimerization) domain of PML were essential for this interaction. Analysis of the effects of PML on Sp1 DNA binding by electrophoretic mobility shift assay (EMSA) showed that PML could specifically disrupt the binding of Sp1 to DNA. Furthermore, cotransfection of PML specifically repressed Sp1, but not the E2F1-mediated activity of the dihydrofolate reductase promoter. Together, these data suggest that the association of PML and Sp1 represents a novel mechanism for negative regulation of EGFR and other Sp1 target promoters.

Characterization of a fusion cDNA (RARA/myl) transcribed from the t(15;17) translocation breakpoint in acute promyelocytic leukemia.

A nonrandom chromosomal translocation breakpoint, t(15;17)(q22;q21), is found in almost all patients with acute promyelocytic leukemia (APL). Most of these breakpoints occur within the second intron of the retinoic acid receptor-alpha (RARA) gene. We screened a cDNA library of APL and have identified and sequenced a cDNA transcribed from the t(15;17) translocation breakpoint. The 5' end of cDNA p1715 consists of 503 bp of the RARA exon II sequence. A 1.76-kb cDNA without homology to any known gene available in GenBank was found truncated downstream. This cDNA sequence was assigned to chromosome 15 by dot blot hybridization of the flow cytometry-sorted chromosomes. We designate this fusion cDNA RARA/myl, which is different from myl/RARA reported by de The et al. (H. de The, C. Chomienne, M. Lanotte, L. Degos, and A. Dejean, Nature (London) 347:558-561, 1990). This result demonstrates that the two different types of hybrid mRNA can be transcribed from this breakpoint. We screened a non-APL cDNA library and identified a 2.8-kb myl cDNA. This cDNA is able to encode a polypeptide with a molecular weight of 78,450. Alternative splicing of the myl gene which resulted in myl proteins with different C terminals was found. Southern blot analysis of the genomic DNA isolated from 17 APL patients by using the myl DNA probe demonstrated that the myl gene in 12 samples was rearranged. Northern (RNA) blot analysis of RARA gene expression in two APL RNA samples showed abnormal mRNA species of 4.2 and 3.2 kb in one patient and of 4.8 and 3.8 kb in another patient; these were in addition to the normal mRNA species of 3.7 and 2.7-kb. The myl DNA probe detected a 2.6-kb abnormal mRNA in addition to the normal mRNA species of 3.2, 4.2, and 5.5 kb. Using the polymerase chain reaction, we demonstrated that both RARA/myl and myl/RARA were coexpressed in samples from three different APL patients. From this study, we conclude that the t(15;17) translocation breakpoint results in the transcription of two different fusion transcripts which are expected to be translated into fusion proteins.

PML, a growth suppressor disrupted in acute promyelocytic leukemia.

The nonrandom chromosomal translocation t(15;17)(q22;q21) in acute promyelocytic leukemia (APL) juxtaposes the genes for retinoic acid receptor alpha (RAR alpha) and the putative zinc finger transcription factor PML. The breakpoint site encodes fusion protein PML-RAR alpha, which is able to form a heterodimer with PML. It was hypothesized that PML-RAR alpha is a dominant negative inhibitor of PML. Inactivation of PML function in APL may play a critical role in APL pathogenesis. Our results demonstrated that PML, but not PML-RAR alpha, is a growth suppressor. This is supported by the following findings: (i) PML suppressed anchorage-independent growth of APL-derived NB4 cells on soft agar and tumorigenicity in nude mice, (ii) PML suppressed the oncogenic transformation of rat embryo fibroblasts by cooperative oncogenes, and (iii) PML suppressed transformation of NIH 3T3 cells by the activated neu oncogene. Cotransfection of PML with PML-RAR alpha resulted in a significant reduction in PML's transformation suppressor function in vivo, indicating that the fusion protein can be a dominant negative inhibitor of PML function in APL cells. This observation was further supported by the finding that cotransfection of PML and PML-RAR alpha resulted in altered normal cellular localization of PML. Our results also demonstrated that PML, but not PML-RAR alpha, is a promoter-specific transcription suppressor. Therefore, we hypothesized that disruption of the PML gene, a growth or transformation suppressor, by the t(15;17) translocation in APL is one of the critical events in leukemogenesis.

The growth suppressor PML represses transcription by functionally and physically interacting with histone deacetylases.

The growth suppressor promyelocytic leukemia protein (PML) is disrupted by the chromosomal translocation t(15;17) in acute promyelocytic leukemia (APL). PML plays a key role in multiple pathways of apoptosis and regulates cell cycle progression. The present study demonstrates that PML represses transcription by functionally and physically interacting with histone deacetylase (HDAC). Transcriptional repression mediated by PML can be inhibited by trichostatin A, a specific inhibitor of HDAC. PML coimmunoprecipitates a significant level of HDAC activity in several cell lines. PML is associated with HDAC in vivo and directly interacts with HDAC in vitro. The fusion protein PML-RARalpha encoded by the t(15;17) breakpoint interacts with HDAC poorly. PML interacts with all three isoforms of HDAC through specific domains, and its expression deacetylates histone H3 in vivo. Together, the results of our study show that PML modulates histone deacetylation and that loss of this function in APL alters chromatin remodeling and gene expression. This event may contribute to the development of leukemia.

Isolation and characterization of six different chicken actin genes.

Genes representing six different actin isoforms were isolated from a chicken genomic library. Cloned actin cDNAs as well as tissue-specific mRNAs enriched in different actin species were used as hybridization probes to group individual actin genomic clones by their relative thermal stability. Restriction maps showed that these actin genes were derived from separate and nonoverlapping regions of genomic DNA. Of the six isolated genes, five included sequences from both the 5' and 3' ends of the actin-coding area. Amino acid sequence analysis from both the NH2- and COOH-terminal regions provided for the unequivocal identification of these genes. The striated isoforms were represented by the isolated alpha-skeletal, alpha-cardiac, and alpha-smooth muscle actin genes. The nonmuscle isoforms included the beta-cytoplasmic actin gene and an actin gene fragment which lacked the 5' coding and flanking sequence; presumably, this region of DNA was removed from this gene during construction of the genomic library. Unexpectedly, a third nonmuscle chicken actin gene was found which resembled the amphibian type 5 actin isoform (J. Vandekerckhove, W. W. Franke, and K. Weber, J. Mol. Biol., 152:413-426). This nonmuscle actin type has not been previously detected in warm-blooded vertebrates. We showed that interspersed, repeated DNA sequences closely flanked the alpha-skeletal, alpha-cardiac, beta-, and type 5-like actin genes. The repeated DNA sequences which surround the alpha-skeletal actin-coding regions were not related to repetitious DNA located on the other actin genes. Analysis of genomic DNA blots showed that the chicken actin multigene family was represented by 8 to 10 separate coding loci. The six isolated actin genes corresponded to 7 of 11 genomic EcoRI fragments. Only the alpha-smooth muscle actin gene was shown to be split by an EcoRI site. Thus, in the chicken genome each actin isoform appeared to be encoded by a single gene.

Immune reactivity of RBL-3, a nonproducer leukemia induced by Rauscher murine leukemia virus in C57BL/6 mice.

A cultured line of RBL-3 leukemia, induced by Rauscher murine leukemia virus (R-MuLV) in C57BL/6 mice, was found to be a virus nonproducer. An NB tropic virus indistinguishable from R-MuLV, however, was recovered after treatment with 5-iodo-2'-deoxyuridine (optimal concentration, 200 microgram/ml). Dexamethasone treatment slightly boosted the production of the activated virus but did not by itself activate the virus. RBL-3 cells were found to have Fc receptors but no surface immunoglobulin or Thy 1.2 antigen. RBL-3 cells exhibited the Friend-Moloney-Rauscher virus-specific transplantation antigen as shown by its immunosensitivity and immunogenicity in tumor rejection experiments. Crude membrane and crude soluble antigen preparations obtained from RBL-3 cell membranes retained the capacity to induce tumor rejection.

Isolation of a B-tropic type-C virus from reticulum cell neoplasms induced in BALB/c mice by SJL/J type-C virus.

D1-murine leukemia virus (MuLV), an N-tropic type-C virus isolated from a spontaneous reticulum cell neoplasm, type B (RCN-B) of an SJL/J mouse was propagated in NIH Swiss mouse embryo cell cultures. When injected into BALB/c mice 1 day after neonatal thymectomy, 30% of the inoculated mice developed RCN-B in 5 months, whereas none of the uninoculated controls did. From the spleen and lymph node extracts of all RCN-B-bearing mice tested, B-tropic type-C viruses (designated E1-MuLV) were isolated in high titers (10-5 minus 10-6 XC plaque-forming units/ml). Only low titers (10-1 minus 10-2 XC plaque-forming units/ml) of N- or B-tropic viruses were isolated from those thymectomized mice, inoculated but nontumorous, whereas only N-tropic viruses were detected in the uninoculated thymectomized mice. No virus was isolated from the nonthymectomized, untreated controls. Antigenically, the viral envelope antigen (VEA) of E1-MuLV was distinct from those of DU-MuLV, xVEA, or Gross-VEA, but some cross reaction with AKR-MuLV-VEA was observed. The relationship of D1-MuLV to E1-MuVL with respect to oncogenesis and viral genome activation was discussed.

Properties of RBL-5 leukemia cells cultivated in vitro.

RBL-5 leukemia induced by Rauscher murine leukemia virus (R-MuLV) carried in the ascites form in C57BL/6 mice was cultivated in vitro and passaged continuously. Both low- and high-passage cells released the same level of murine leukemia virus, as detected by the XC plaque assay. Membrane antigens of the cultivated cells were also detected by the indirect immunofluorescence assay. Both low- and high-passage cells showed the same activity of antigen detectable by anti-R-MuLV serum, whereas only the high-passage cells reacted strongly against a syngeneic antiserum to RBL-5 crude membrane (CM). The oncogenicity of the low and high passages of the cultivated cells was compared with the ascites cells. The results indicated that the oncogenic potential of the cells gradually decreased during in vitro passage. The accumulation or retention of CM antigen on the membrane of the high-passage cells suggested that their decreased oncogenic potential might be due to increased immunogenicity and/or immunosensitivity. This strengthened the hypothesis that the cellular transplantation antigens are not identical to type C viral antigens, since the R-MuLV antigens were not altered during passage in vitro.

Performance estimation of a Venturi scrubber using a computational model for capturing dust particles with liquid spray.

A Venturi scrubber has dispersed three-phase flow of gas, dust, and liquid. Atomization of a liquid jet and interaction between the phases has a large effect on the performance of Venturi scrubbers. In this study, a computational model for the interactive three-phase flow in a Venturi scrubber has been developed to estimate pressure drop and collection efficiency. The Eulerian-Lagrangian method is used to solve the model numerically. Gas flow is solved using the Eulerian approach by using the Navier-Stokes equations, and the motion of dust and liquid droplets, described by the Basset-Boussinesq-Oseen (B-B-O) equation, is solved using the Lagrangian approach. This model includes interaction between gas and droplets, atomization of a liquid jet, droplet deformation, breakup and collision of droplets, and capture of dust by droplets. A circular Pease-Anthony Venturi scrubber was simulated numerically with this new model. The numerical results were compared with earlier experimental data for pressure drop and collection efficiency, and gave good agreements.

Susceptibility of xeroderma pigmentosum cells to transformation by murine and feline sarcoma viruses.

Among various strains of skin fibroblasts tested, two strains derived from xeroderma pigmentosum (XP) patients (ages 19 and 25) with neurological complications and two strains obtained from heterozygotes (ages 54 and 18) showed relatively higher susceptibility than normal age-matched controls to transformation by feline sarcoma virus (FSV). Only one strain from a normal individual also showed a high susceptibility. Generally, there was a parallelism in susceptibilities to FSV and Kirsten murine sarcoma virus (KiMsv). However, cells from normal individuals of 46 years or older exhibited high ratios of FSV:KiMSV titers which were due to their lower susceptibility to KiMSV. Cells from two XP patients (ages 25 and 22) and a heterozygote (age 18), who were in a younger age group, manifested such a differential susceptibility to FSV and KiMSV. There was a correlation between the relative sensitivity of XP cells to the cytotoxic effect of 4-nitroquinoline 1-oxide and killing effect of UV light. Pretreatment of fibroblasts from three XP patients by a subtoxic dose of 4-nitroquinoline 1-oxide 24 hr before viral infection facilitated transformation by KiMSV and FSV, whereas no such effect was observed with three normal cells strains similarly treated.

Amplification and enhanced expression of the c-Ki-ras2 protooncogene in human embryonal carcinomas.

Two cell lines of human embryonal carcinoma, Tera-1 and Tera-2, have been found to exhibit a 4- to 6-fold amplification of protooncogene c-Ki-ras2. The polyadenylic acid selected RNA also showed 8-fold or greater enhancement, showing marked elevation in the level of two major mRNAs, 5.7 and 4.0 kilobases, and two additional minor mRNAs, 2.3 and 1.2 kilobases, as compared with those of a normal human embryonic fibroblast cell line, MRC-5. More than one-half of the number of tumor samples obtained from metastatic human embryonal carcinomas also showed c-Ki-ras2 gene amplification and enhanced mRNA expression. However, the c-Ki-ras2 gene amplification did not always lead to enhanced mRNA expression, and some embryonal carcinomas showed mRNA overexpression without apparent c-Ki-ras2 gene amplification. These results suggest that human embryonal carcinomas may have c-Ki-ras2 amplification and/or overexpression before in vitro culture. Among various chromosomal changes observed in Tera-1 and Tera-2 cells, there were anomalies in chromosome 12 in which c-Ki-ras2 is located although these karyological changes alone could not account for the amplification observed. It is suggested that the genomic instability and active DNA replication during the early developmental period may give rise to changes involving c-Ki-ras2 which may contribute to oncogenic processes.

Adenovirus-mediated expression of PML suppresses growth and tumorigenicity of prostate cancer cells.

Our previous studies demonstrated that the promyelocytic leukemia gene, PML, encodes a growth and transformation suppressor. Overexpression of PML inhibits cancer cell growth in vitro and in vivo. In this study, we further explored the possibility of applying PML as a potential agent for developing prostate cancer gene therapy using an adenovirus delivery system. We have constructed and produced the recombinant PML-adenovirus, Ad-PML, in which the full-length PML cDNA is driven by the strong cytomegalovirus promoter. In LNCaP, DU145, and PC-3 prostate cancer cell lines, an infection efficiency of 90% can be achieved at a concentration of 2, 10, and 100 multiplicity of infection (MOI), respectively. Western blotting and immunofluorescence staining demonstrated that the AD-PML-infected cells expressed a high level of PML protein. The protein expression peaked at days 3-4 postinfection, and a detectable level of PML was found at day 18 after viral infection. To test the effect of Ad-PML on the growth of prostate cancer cells, the DU145 and LNCaP cells were infected with 10 and 2 MOI of Ad-PML. We found that the growth rate of the Ad-PML-infected DU145 and LNCaP cells were significantly inhibited. A tumorigenicity test in nude mice showed that the Ad-PML-treated DU145 cells failed to form tumors. Most importantly, direct injection of Ad-PML into DU145-induced tumors was able to repress tumor growth in nude mice by 64%. Taken together, these data indicate that PML is a tumor growth suppressor in prostate cancer and that Ad-PML may be a potential candidate for human prostate cancer therapy.