PolyI.polyC12U-mediated inhibition of loss of alloantigen responsiveness viral replication in human CD4+ T cell clones exposed to human immunodeficiency virus in vitro.

Two alloreactive human CD4+ T cell clones, recognizing HLA-DR2 and HLA-DR1 determinants, lost their specific proliferative capacity after infection with HIV. This system was used to explore the effect of polyI.polyC12U on HIV replication and immune suppression. The mismatched double-stranded RNA blocked HIV-associated particulate reverse transcriptase activity and viral-mediated cytopathic effects. Also, polyI.polyC12U preserved the alloreactivity of T cell clones after exposure to HIV.PolyI.polyC12U appeared to act at a level subsequent to host cell infection and reverse transcription. It had no effect on the enhancement of gene expression by the HIV transcription unit tatIII. These findings indicate that early in the course of infection of CD4+ T lymphocytes, HIV can directly abrogate proliferation to specific allodeterminants, and that this function is preserved in the presence of polyI.polyC12U. They also provide insight into the mechanism of antiviral action of a class of agent with potential clinical utility in AIDS.

Neoplastic conversion of preneoplastic Syrian hamster cells: rate estimation by fluctuation analysis.

Analysis of the role of gene mutations in the multistep process of neoplastic transformation requires that the discrete steps in carcinogenesis first be dissected. Toward this end, we have isolated and characterized preneoplastic Syrian hamster cells which exhibit in vitro a trait highly correlated with neoplastic conversion in vivo. Previous findings (J. C. Barrett, Cancer Res. 40:91-94, 1980) indicate that spontaneous neoplastic transformation of Syrian hamster cells occurs in at least two steps. An intermediate stage, characterized by an aneuploid established cell line which has a propensity to become neoplastic spontaneously upon further growth in vitro, has been described. These preneoplastic cells differ from diploid early-passage Syrian hamster cells in becoming capable of anchorage-independent growth in semisolid agar, as well as becoming neoplastic in vivo when attached to a solid substrate. Evidence presented here demonstrates that anchorage-independent conversion in vitro is a reliable marker for neoplastic conversion in this cell system. Fluctuation analyses, patterned after those described by Luria and Delbruck for microbial genetics, demonstrate that anchorage-independent variants are generated randomly from clonally derived preneoplastic cells at the rate of 10(-8) to 10(-7) variants per cell per generation. These results establish a multistep stochastic process for transformation in vitro and indicate that conversion to anchorage independence may be necessary for Syrian hamster cells to become tumorigenic. The possible role of gene mutation in this step during neoplastic progression is discussed.

Requirement of long progression time for the expression of neoplastic phenotypes following direct perturbation to specific region(s) of DNA of Syrian hamster embryo cells.

We have studied the neoplastic transformation of early passage Syrian hamster embryo cells in culture, which were initiated by a specific perturbation of DNA replicated during different periods of S phase. The cells were treated with a "1-h pulse" of 5-bromodeoxyuridine (BrdUrd), followed by a "5-min" irradiation with near ultraviolet (near UV) light. The treated cells were successively subcultured and tested for growth in soft agar and tumorigenicity in newborn hamsters. Neoplastic transformation was induced by treatment with BrdUrd followed by near UV irradiation within the first 4 h of S phase, which was 5 h in duration. Cells treated with BrdUrd plus near UV irradiation during late S phase (the last hour) or during the absence of DNA synthesis (outside of the S phase) were not transformed. In addition, cultures treated in either G1, G2, or S phase with BrdUrd alone or with near UV alone also were not transformed. While morphological transformation, somatic mutation, and DNA and chromosomal aberrations can be observed either immediately or after a few cell divisions, 100 to 200 population doublings after the treatment were required for the emergence of a neoplastic subpopulation, as measured by anchorage independent growth and tumorigenicity. Furthermore, cellular DNA replicated during the first 4 h of S phase is sensitive to the perturbation which leads to neoplastic transformation. These results indicate that the direct perturbation of specific region(s) of cellular DNA can initiate the neoplastic transformation process; but for full expression of the neoplastic phenotypes, a long progression time is required for the acquisition of anchorage-independent growth and tumorigenicity.

DNA replication in Syrian hamster cells transiently exposed to hydroxyurea.

DNA rereplication has been observed in mammalian cells transiently treated during S phase with hydroxyurea, an inhibitor of DNA synthesis. Recently, evidence has been presented which does not support this proposal. We have performed a series of similar but more defined studies with the tumorigenic Syrian hamster cell line BP6T to further investigate the possibility of DNA rereplication after transient inhibition during S phase. Synchronized BP6T cells (greater than 75%) were given a 6-h exposure to 1 mM hydroxyurea after having progressed 2 h into S phase. The DNA species synthesized up to 24 h after removal of the inhibitor have been identified by bromodeoxyuridine pulse labeling and density gradient centrifugation studies. The results indicate that no DNA rereplication had occurred in the S phase of the same cell cycle as the transient hydroxyurea treatment. DNA replicated early in the S phase in which treatment was given was not replicated again until the next cell cycle. Our observations reveal that DNA rereplication does not occur in tumorigenic Syrian hamster cells transiently insulted with hydroxyurea during S phase. Thus, these findings imply that DNA synthesis is stringently controlled in these transformed cells.

Quantitation of fibrinolytic activity of Syrian hamster fibroblasts using 3H-labeled fibrinogen prepared by reductive alkylation.

Tritium-labeled fibrinogen with a specific activity of 2.0 X 10(7) cpm/mg was prepared by the method of reductive alkylation. The use of the 3H-fibrinogen as a substrate for detection of both intracellular and extracellular fibrinolytic activity derived from cultures of benzo(a)pyrene-transformed Syrian hamster cell lines was examined in cell-free assays, 3H-fibrinogen enabled reliable quantitation of the fibrinolytic activity associated with neoplastic cells. The elevated extracellular fibrinolytic activity in the transformed cell lines as compared to normal hamster embryo cultures was demonstrated with this substrate. The ease with which large quantities of 3H-fibrinogen of high specific activity and prolonged half-life can be prepared makes the use of this substrate an attractive alternative to 125I-labeled fibrinogen.

Toxicity of metabolic benzo(a)pyrenediones to cultured cells and the dependence upon molecular oxygen.

The three quinone metabolites of carcinogenic benzo(a)pyrene, the isomeric benzo(a)pyrenediones (6, 12; 1,6; 3,6), are toxic to cultured hamster cells at low concentrations. The reduction in cell number, observed after treatment with these metabolites, is the result of both direct cell killing and the inhibition of growth, since DNA synthesis is inhibited very early after treatment with benzo(a)pyrene 1,6-dione when little cell death has occurred. The rate of RNA synthesis was also inhibited by treatment of cells with benzo(a)pyrene 3,6-dione. These actions of the benzo(a)pyrenediones toward hamster cells can be eliminated or substantially reduced by the removal of oxygen from the growth medium and atmosphere in which the cells are incubated. In contrast, anaerobic conditions do not reduce the cytotoxicity observed with the alkylating agent ethyl methanesulfonate. These results support the hypothesis that benzo(a)pyrenediones, and other biologically active quinones, owe their activity to oxidation-reduction cycles involving quinone, hydroquinone, and molecular oxygen; the reactive reduced oxygen radicals and semiquinone radical formed during these cycles may be responsible for the observed cellular injury and inhibition of cellular processes.

A qualitative and quantitative assay for cells lacking postconfluence inhibition of cell division: characterization of this phenotype in carcinogen-treated Syrian hamster embryo cells in culture.

We have developed a qualitative and quantitative assay system for detecting cells lacking postconfluence inhibition of cell division (contact insensitivity, CS-) in golden Syrian hamster embryo cells in culture by measuring the number of cells able to form colonies on a lethally irradiated, confluent monolayer of a contact-sensitive established cell line. A subpopulation in normal low-passage cultures of golden Syrian hamster embryo cells temporarily exhibits this CS- phenotype at very low frequency (approximately 4 x 10(-3)) but quickly loses the property within a few passages in vitro. This phenotype is invariably exhibited by various tumorigenic cell lines at very high frequency (7 to 50 x 10(-2)) and appears to correlate with the anchorage-independent growth phenotype. The temporal acquisition of the CS- phenotype by tertiary-passage golden Syrian hamster embryo cells following exposure to N-methyl-N'-nitro-N-nitrosoguanidine was examined. Cells with a stably heritable CS- phenotype are detected after approximately 20 posttreatment population doublings. In contrast, anchorage-independent cells are not detected until 35 to 95 posttreatment population doublings. These CS- cells appear to be preneoplastic cells, since clonally isolated CS- cells did not exhibit anchorage-independent growth until further passaging in vitro. The results suggest that acquisition of the CS- phenotype represents an early stage in neoplastic progression.

Correlation of in vitro growth properties and tumorigenicity of Syrian hamster cell lines.

Several in vitro phenotypic characteristics frequently associated with neoplastic cells were examined in a series of spontaneous and benzo(a)pyrene-induced Syrian hamster clonal cell lines which differed in their degree of tumorigenicity. Nonparametric statistical analysis demonstrated cloning efficiency in semisolid agar, enhanced fibrinolytic activity, decreased serum requirement for growth, decreased organization of intracellular actin, and increased cloning efficiency in liquid medium to be correlated with tumorigenicity. These correlations were not only qualitative but also quantitative. This suggests that the factors determining the degree of tumorigenicity of a cell can be cellular growth properties.

Temporal acquistion of enhanced fibrinolytic activity by syrian hamster embryo cells following treatment with benzo(a)pyrene.

Following treatment of Syrian hamster embryo cells with benzo(a)pyrene, the time required for the expression of enhanced fibrinolytic activity was examined. For this study, the fibrin-agarose overlay method was developed to distinguish the activity of normal and transformed colonies of hamster cells. Colonies possessing enhanced fibrinolytic activity were not observed one passage (2 weeks after treatment). Morphologically transformed colonies, which exhibited no enhanced fibrinolytic activity, were observed 8 days following treatment. In contrast to these two early changes, cells capable of growth in soft agar were observed much later (6 to 8 weeks after treatment). Untreated Syrian hamster embryo cells generally senesced and did not exhibit enhanced fibrinolytic activity. Approximately 1 of 10 untreated cultures escaped senescence and evolved as a continuous cell line; such cultures frequently exhibited enhanced fibrinolytic activity. These results suggest that the acquisition of enhanced fibrinolytic activity, while perhaps not a cause of neoplastic transformation, may reflect a loss of control of the normal function of the cellular genetic apparatus during the process of transformation.

A simple method of the preparation of 2'-O-methyladenosine. Methylation of adenosine with methyl iodide in anhydrous alkaline medium.

A simple and effective method of the methylation on the 2'-O position of adenosine is described. Adenosine is treated with CH3I in an anhydrous alkaline medium at 0 degrees C for 4 h. The major products of this reaction are monomethylated adenosine at either the 2'-O or 3'-O position (total of 64%) and the side products are dimethylated adenosine ((2', 3'-O-dimethyladenosine, 21%, and N6-2'-O-dimethyladenosine, 11%). The ratio of 2'-O- and 3'-O-methyladenosine has been found to be 8 to 1. Therefore, this reaction preferentially favors the synthesis of 2'-O-methyladenosine. The monomethylated adenosine is isolated from reaction mixture by a silica gel column chromatography. Then the pure 2'-O-methyladenosine can be separated by crystallization in ethanol from the mixture of 2'=O and 3'-O-methylated isomers. The overall yield of 2'-O-methyladenosine is 42%.

The attachment and penetration of T7 DNA/phage in Syrian hamster embryonic cells.

Bacteriophage T7 DNA can penetrate Syrian hamster embryonic cells after a mandatory initial pretreatment with DEAE-dextran. In 3 h an extracellular complex between T7DNA and the cell monolayer is formed which is equivalent to 105 T7 genomes per cell. During the ensuing 24-48 h of cell growth, an average of 102-103 T7 genomes are transported to the nucleus in 90% of the cells of the culture.

In vitro senescence of Syrian hamster mesenchymal cells of fetal to aged adult origin. Inverse relationship between in vivo donor age and in vitro proliferative capacity.

Normal diploid Syrian hamster dermal mesenchymal cell strains, regardless of the age of the tissue of origin, exhibit in vitro cellular senescence. The frequency of spontaneous escape from senescence and conversion to a permanent cell line is less than 5% among replicate flasks. The overall pattern of senescence of cells of fetal, neonatal, young adult (6 months) and aged adult (24 months) origin is similar in terms of the morphological changes and proliferative changes indicated by the reduction of saturation density, cloning efficiency and [3H]thymidine labeling index and by the increase in population doubling time and cell volume. However, the average maximum cumulative population doubling level is characteristic for each cell type: 13-day gestation fetal cells, 28.6; neonatal cells, 18.7; young adult cells, 13.8; aged adult cells, 11.1. Thus, the in vitro proliferative capacity of Syrian hamster mesenchymal cells is inversely related to the in vivo age of the donor.

Control of ribonucleic acid function by oligonucleoside methylphosphonates.

Oligodeoxyribonucleoside methylphosphonates contain nonionic 3'-5' linked methylphosphonate internucleotide bonds in place of the normal charged phosphodiester linkage of natural nucleic acids. These oligomers are resistant to nuclease hydrolysis, can pass through the membranes of mammalian cells in culture and can form stable hydrogen-bonded complexes with complementary nucleotide sequences of cellular RNAs such as mRNA. The oligomers are readily synthesized on insoluble polymer supports. Their chainlength and nucleotide sequence can be determined by chemical sequencing procedures. Oligonucleoside methylphosphonates which are complementary to the 5'-end, initiation codon region, or coding region of rabbit globin mRNA inhibit translation of the mRNA in rabbit reticulocyte lysates and globin synthesis in rabbit reticulocytes. This inhibition is due to the interaction of the oligomers with mRNA and the extent of inhibition is influenced by the secondary structure of the mRNA and the location of oligomer binding site on the mRNA. Oligomers complementary to the initiation codon regions of N, NS and G protein mRNAs of Vesicular stomatitis virus (VSV) inhibit virus protein synthesis in VSV-infected Mouse L-cells. These oligomers do not affect L-cell protein synthesis or growth. Virus protein synthesis and growth can also be selectively inhibited by oligonucleoside methylphosphonates which are complementary to the donor or acceptor splice junctions of virus pre mRNA. An oligomer complementary to the donor splice junction of SV40 large T antigen mRNA inhibits T-antigen synthesis in SV40-infected African green monkey kidney cells but does not inhibit overall cellular protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Simultaneous in situ detection of beta-interferon mRNA and DNA in the same cell.

We report a quantitative method that combines in situ mRNA hybridization with microfluorometric analysis of DNA content to detect gene expression in single cells of a heteroploid cell population. The model was a human fibrosarcoma HT1080 cell line which consisted of diploid and tetraploid cells that were induced with polyI:polyC for production of beta-interferon. The level of beta-interferon mRNA detected by in situ hybridization was found to be two to three times higher in tetraploid compared to diploid HT1080 cells, and correlated with beta-interferon activity in that a subclone of tetraploid HT1080 cells secreted two- to fivefold more beta-interferon than a subclone of diploid HT1080 cells. Interestingly, beta-interferon-related transcripts were detected during S-phase in uninduced tetraploid HT1080 cells. In addition, beta-interferon induced by polyI:polyC was expressed in all phases of the cell cycle as demonstrated with a human diploid fibroblast, HF926. The unique features offered by the combination of microfluorometry and in situ hybridization provide a valuable tool to investigate specific gene expression related to ploidy or cell-cycle stage in the same individual cell of an unsynchronized population. Since the method allows direct observation of morphology, one can be assured that all quantitative measurements were made on whole cells with intact nuclei.

Antiviral effect of oligo(nucleoside methylphosphonates) complementary to the herpes simplex virus type 1 immediate early mRNAs 4 and 5.

We have previously shown that an oligo(nucleoside methylphosphonate) (deoxynucleoside methylphosphonate residues in italics) complementary to the acceptor splice junction of herpes simplex virus type 1 (HSV-1) immediate-early (IE) pre-mRNAs 4,5 [d(TpTCCTCCTGCGG)], causes sequence-specific inhibition of virus growth in infected cell cultures (Smith et al., 1986; Kulka et al., 1989). Here we report a similar inhibition of HSV-1 growth by oligo(nucleoside methylphosphonates) complementary to the splice donor site of HSV-1 IE pre-mRNAs 4,5 [d(GpCTTACCCGTGC)] and to the translation initiation site of IE4 mRNA [d(ApATGTCGGCCAT)]. An oligomer complementary to the translation initiation site of IE5 mRNA [d(GpGCCCACGACAT)] or an unrelated oligomer [d(GpCGGGAAGGCAC)] did not inhibit virus growth. IC50 values were 20, 25 and 20 microM for d(TpTCCTCCTGCGG), d(GpCTTACCCGTGC) and d(ApATGTCGGCCAT) respectively. In infected BALB/c mice d(TpTCCTCCTGCGG) caused a significant decrease in HSV-1 growth (82% inhibition at 500 microM). A psoralen-derivative of d(TpTCCTCCTGCGG) that binds covalently to complementary sequences after exposure to 365 nm irradiation, inhibited HSV-1 growth (86-91%) at a 10-fold lower concentration than the non-derivatized oligomer. The inhibition was sequence-specific and significantly lower (27%) for HSV-2 that differs from HSV-1 in 7 of the 12 bases targeted by d(TpTCCTCCTGCGG). Virus growth was not inhibited by d(GpGCCCACGACAT). The data suggest that oligo(nucleoside methylphosphonates) may be effective antiviral agents.

A sensitive assay for the IFN-regulated 2-5A synthetase enzyme.

2-5A synthetase is the central enzyme of the 2-5A system, an important mediator of interferon action. An assay capable of detecting low, yet biologically important levels of 2-5A synthetase enzyme activity is described. The purification of enzyme reaction products on SepPak C-18 cartridges resulted in a significant reduction in background, when a high specific activity substrate was used to label the 2-5A. Quantitation of labeled 2-5A by chromatography and scintillation counting provided a means of detecting femptomolar amounts of 2-5A. The combination of these procedures accounts for a 3-4 log increase in sensitivity over existing assays. This degree of sensitivity should permit a more accurate determination of the 2-5A synthetase activity in vivo leading to a better understanding of the role of the 2-5A system in virus infection and other cellular processes.

Detection of intact prostate cancer cells in the blood of men with prostate cancer.

OBJECTIVES: To develop a procedure to be used to find, identify, and characterize the living prostate cancer cells in the blood of patients with prostate cancer. METHODS: The procedure is based on a negative selection approach that removes most of the blood cells and collects the remaining prostate cancer cells, which are identified and characterized by fluorescent in situ hybridization with deoxyribonucleic acid probes and by indirect fluorescent immunocytochemical staining. The blood cells are removed via density gradient centrifugation. RESULTS: Using the prostate cancer LNCaP cells as a model, the recovery rate of the added prostate cancer cells to 10 mL of blood was about 85%, with a dilution of 1 LNCaP cell to 10,000 white blood cells or more. Blood samples varying from 9 to 27 mL were collected and analyzed from 8 men aged 54 to 79 years who had varying levels of PSA in serum. In one blood sample, prostate cancer cells were not found; in the seven other samples, the number of prostate cancer cells found per milliliter of blood varied from 1 to 20. Prostate cancer cells were not found in 7.5 to 15-mL blood samples from 3 healthy younger men. The prostate cells were found to be aneuploid for chromosomes 7 and 8, highly suggestive that these cells were cancerous. CONCLUSIONS: Using a negative selection approach, prostate cells can be found in the blood of patients with prostate cancer, as identified by prostate cell-specific probes and antibodies. These cells were found to be aneuploid.

Epidermal growth factor-urogastrone action: induction of 2',5'-oligoadenylate synthetase activity and enhancement of the mitogenic effect by anti-interferon antibody.

Epidermal growth factor-urogastrone (EGF-URO), early in the course of stimulating DNA synthesis in quiescent human fibroblasts, also causes a three to five-fold elevation of the activity of intracellular 2',5'-oligoadenylate synthetase (2,5A synthetase), an enzyme that has been previously implicated in the antiproliferative effects of interferon. The increase in synthetase activity precedes DNA synthesis by approximately six hours, with maximal synthetase activity either preceding or coinciding with maximal DNA synthesis. EGF-URO stimulation does not result in the secretion of detectable amounts of interferon (IFN) into the growth medium and anti-human IFN-beta antibodies do not block the EGF-URO-mediated rise in 2,5A synthetase activity. Thus, the elevation of 2,5A synthetase can be attributed to the action of EGF-URO itself, and not to IFN. Nonetheless, in the presence of anti-human interferon-antibodies, the time course of EGF-URO-stimulated DNA synthesis is prolonged both in human and in Syrian hamster embryo fibroblasts; the effects of the antibody were reversed in both cell strains by the addition of human IFN-beta (HuIFN-beta). The data suggest a role for 2',5'-oligoadenylate synthetase in the process of EGF-URO-mediated mitogenesis and point to the possible production of interferon-related cell-associated regulators during the course of EGF-URO action.

Epitopic discrimination by monoclonal antibodies directed against the same alkylated nucleoside.

Epitopic specificity of three monoclonal antibodies (mAb's) (coded as ER-6, ER-3, and EM-1) was examined through the utilization of haptenic structural analogs. The binding affinity expressed by the microscopic equilibrium constant (Ki) (Yuhasz, et al., Biochemistry 26, 2334-2342 (1987] of the immunizing hapten, O6-ethyl-2'-deoxy-guanosine (*G) and eight structural analogs, were analyzed by a nitrocellulose affinity filter assay (NAFA) and radioimmunoassay (RIA) for each mAb to determine the protein-hapten interaction between the epitope and the binding cavity. Several components of the *G hapten were determined to be critical for each mAb recognition, while all three mAb's were found to require the O6-ethyl moiety, conjugated guanine base ring, the glycosyl bond and the sugar ring C [1'] and C [2'] position. This investigation further probes and categorizes the binding specificity of the monoclonal antibodies after incorporation of the *G monomer into three short deoxyribooligomeric haptens: O6-ethyl-2'-deoxyguanylyl 3',5' deoxyadenosine (*GA), 2'-deoxyadenylyl 3',5' O6-ethyl-2'-deoxyguanylyl 3',5' 2'-deoxyadenosine (A*GA), and O6-ethyl-2'-deoxyguanylyl 3',5' 2'-deoxyadenylyl 3',5'-2'-deoxyadenylyl 3',5' 2'-deoxycytosine (*GAAC). Unlike the similar binding profiles for the monoclonal antibodies and the haptenic structural analogs, the binding profiles for the deoxyribooligomeric haptens were found to differ in their modes of recognition. These results will be compared to ascertain the key components of monomer and oligomer interaction of the binding cavity. It is important for investigations where monoclonal antibodies derived from small haptens are utilized in recognition of larger antigens containing those haptens.

Phosphate catalyzed exchange rates of NH-N protons of DNA.

We have studied the catalysis of the exchange of the hydrogen-bonded NH-N protons of the short DNA helix (d-CCAAGCTTGG)2 by phosphate addition. The NH exchange rates were monitored by the line widths of the corresponding NH resonances in the 1H nmr spectra. The exchange catalyst phosphate is most effective on the exchange rate of the terminal CG1 base pairs. However, all internal base pairs are also moderately affected by phosphate which suggests an exchange mechanism governed by a fast equilibrium between opened and closed states of the duplex. Within the limits of error the same effectiveness of phosphate on the exchange rate of all internal NH-N protons has been observed. With the exception of the terminal base pairs, no sequence and/or position specificity of the exchange rates of the NH-N protons of the base pairs has been found.