Apoptosis induction in prostate cancer cells by a novel gene product, pHyde, involves caspase-3.

A novel gene, pHyde, was recently cloned from Dunning rat prostate cancer cells. A recombinant adenovirus containing pHyde cDNA gene (AdpHyde) was generated to investigate the biological function of pHyde protein. AdpHyde inhibited the growth of human prostate cancer cells. Apoptosis was induced in AdpHyde transduced cells as demonstrated by DAPI (4', 6-diamino-2-phenylindole), TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick and labeling) staining, and flow cytometry assays. Apoptosis was also induced in human xenograft prostate cancer tumors growing in nude mice following treatment with AdpHyde. AdpHyde transduction resulted in a dose-dependent stimulation of caspase-3 activity in DU145 cells which was blocked by DEVD (succinyl-Asp-Glu-Val-Asp-aldehyde) and VAD (benzyloxycarbonyl - Val - Ala - Asp -fluoromethylketone), inhibitors specifically against caspase-3. Moreover, cancer cells that lacked expression of endogenous caspase-3 were not or barely inhibited by pHyde. These results taken together suggest that pHyde inhibits cancer growth by inducing apoptosis through a caspase-3 dependent pathway.

Role of pHyde novel gene product as an intrinsic factor for apoptotic pathway in prostate cancer.

Induction of apoptotic cell death mechanism can be regulated by internal factor(s), such as by gene product(s) that directly upregulate the apoptosis pathway or indirectly by down-regulating the anti-apoptosis gene. This homeostasis is a normal phenomenon in a biological system disturbed by cancer. It is thus important to find any gene functioning as an upregulator for the apoptosis pathway that may have a potential application in the context of cancer gene therapy. We have cloned a novel rat gene, denoted as pHyde, that fulfilled this objective. Internally, this pHyde gene product renders the stable transfectant of rat prostatic cancer cell lines more susceptible to apoptosis even without any external inducer. By using an external agent, such as 5-fluoro-2'-deoxyuridine (FdUr), apoptotic responses of the stable transfectants are even higher, suggesting that both intrinsic and extrinsic factors work synergistically. The pHyde gene product was termed an intrinsic factor, whereas FdUr was considered an extrinsic factor for the apoptosis in rat prostate cancer model.

Application of an improved cDNA competition technique to identify prostate cancer-associated gene.

A technique to improve cDNA library screening was developed by using mixed probes derived from two closely related cDNA populations of high-metastatic MAT-LyLu and low-metastatic AT-1 Dunning R3227 rat prostate cancer sublines. The technique required the generation of a cDNA library from each subline followed by polymerase chain reaction (PCR) amplification of the cDNA insert population. The PCR products derived from the first library were radiolabeled and mixed with an excess amount of PCR products from the second library. The mixture and an excess amount of both the lambda and pBluescript DNA were used as a probe to screen the first cDNA library. This mixed probe (designated the competition probe) differentially cross-hybridized with the plaque lift of the screened first cDNA library. Weak radioactive signals indicated the cross-hybridization of cDNA sequences common to the competition probe mixture and the first cDNA library, whereas strong signals implied unhybridized unique or abundant cDNA sequences in the first cDNA library. The reproducibility of this technique was confirmed by showing that the full-length cDNA clones were associated with the phenotype of the screened first cell line. The isolated clones were characterized as rat nucleolar protein, rat mitochondrial genes coding for 16S and 12S rRNAs, and rat tRNAs specific for valine and phenyl-alanine. This result is consistent with the fact that the first cell line, MAT-LyLu, is metabolically more active than are AT-1 cells because of higher gene dosage or amplification of nucleolar and mitochondrial RNA and its associated genes. Another clone which had a strong signal represented a novel gene associated with the MAT-LyLu cancer phenotype.

Cloning, characterization, and functional studies of a nonintegrin platelet receptor for type I collagen.

A cDNA (1.6 kb) encoding a platelet protein receptor that binds type I collagen has been isolated from a human bone marrow cDNA library by using a degenerate oligonucleotide probe derived from the amino acid sequence of a CNBr fragment of the purified receptor. Computer search revealed that this cDNA represents the coding sequence of a unique protein. Using the prokaryotic expression system pKK 223-3-65 cDNA, a 54-kD recombinant protein was obtained and purified to apparent homogeneity. In an eukaryotic expression vector (pcDNA3-65 cDNA), a 65-kD protein was identified that was recognized by monoclonal anti-65 kD antibody (anti-65m). The recombinant protein binds to type I, but not to type III collagen by affinity column chromatography. The binding of the recombinant protein to type I collagen-coated Petri dishes is inhibited by anti-65m in a dose-dependent manner. The pcDNA3-65 cDNA-transfected nonadherent T cells express the protein, allowing them to attach to a type I collagen matrix, and are inhibited by anti-65m in a dose-dependent manner. Like the receptor protein purified from platelet membranes, the recombinant protein inhibits type I collagen-induced platelet aggregation and the adhesion of [14C]serotonin-labeled platelets to type I collagen in a dose-dependent manner. The recombinant protein neither binds to type III collagen-coated Petri dishes nor inhibits type III collagen and ADP-induced platelet aggregation, indicating specificity for type I collagen.

A xeroderma pigmentosum complementation group A related gene: confirmation using monoclonal antibodies against the cyclobutane dimer and (6-4) photoproduct.

Xeroderma pigmentosum complementation group A was partially complemented by a cosmid genomic clone containing a 42-kb human DNA insert selected with a cDNA clone that we obtained through cDNA competition between the repair-proficient and repair-deficient cell line. The relationship between these two clones was confirmed using PCR amplifications. The enhancement in DNA-repair capacity of the transformants was assessed with the monoclonal antibodies specific for cyclobutane dimers and (6-4) photoproducts and partially correct the xeroderma pigmentosum complementation group A defect. Furthermore, the level of the photoproduct-repair capacity is in agreement with the survival enhancement calculated from the D37 values. This gene was mapped to chromosome 8, suggesting that this may represent one of the defective gene(s) in xeroderma pigmentosum complementation group A.

A gene that partially complements xeroderma pigmentosum group A cells maps to human chromosome 8.

A gene that partially complements sensitivity of xeroderma pigmentosum cells of group A to UV irradiation has been mapped to human chromosome 8. Isolation of this gene has previously been described. A cDNA clone pEMKR that represents part of this gene was used for mapping. Based upon the nucleotide sequence of pEMKR, a set of oligonucleotide primers were designed for PCR amplification of DNAs from hybrid cell lines. A panel of rodent-human hybrid cell lines representing the total human genome was screened by PCR and Southern blot analysis for chromosomal assignment of this gene. PCR amplification and hybridization occurred only in the case of human and hybrid cell lines that contained human chromosome 8. The pEMKR thus represents a different gene than a DNA repair gene XPAC that has been mapped to human chromosome 9.

Increased UV resistance in xeroderma pigmentosum group A cells after transformation with a human genomic DNA clone.

Xeroderma pigmentosum (XP) is an autosomal recessive disease in which the major clinical manifestation is a 2,000-fold enhanced probability of developing sunlight-induced skin tumors, and the molecular basis for the disease is a defective DNA excision repair system. To clone the gene defective in XP complementation group A (XP-A), cDNA clones were isolated by a competition hybridization strategy in which the corresponding mRNAs were more abundant in cells of the obligately heterozygous parents relative to cells of the homozygous proband affected with the disease. In this report, a human genomic DNA clone that contains this cDNA was transformed into two independent homozygous XP-A cell lines, and these transformants displayed partial restoration of resistance to the killing effects of UV irradiation. The abundance of mRNA corresponding to this cDNA appears to correlate well with the observed UV cell survival. The results of unscheduled DNA synthesis after UV exposure indicate that the transformed cells are repair proficient relative to that of the control XP-A cells. However, using this same genomic DNA, transformation of an XP-F cell line did not confer any enhancement of UV survival or promote unscheduled DNA synthesis after UV exposure.

Gene cloning using cDNA libraries in a differential competition hybridization strategy: application to cloning XP-A related genes.

A competition hybridization strategy using size-cut cDNA libraries as both probe and competitor was designed for the cloning of genes whose mRNAs are either regulated transcriptionally or vary in abundance as a function of cell line or cell cycle. We used this strategy to construct cDNA libraries from a particular size fraction of mRNA from three members of a xeroderma pigmentosum (XP), complementation group A family. Size-cut cDNA libraries derived from the father's, mother's, and child's fibroblast cell line were used in a competition scheme to screen two lambda gt11 human cDNA libraries. Of the 15 positive lambda gt11 clones which have been characterized, at least 14 clones represent the same gene which is present in greater abundance in both the mother and father XP obligate heterozygotes relative to the homozygous affected child.

Change in antigen specificity of cytotoxic T lymphocytes is associated with the rearrangement and expression of a T-cell receptor beta-chain gene.

Cloned H-Y-specific murine cytotoxic T lymphocytes, which alter antigen specificity in vitro ("aging"), simultaneously exhibit changes in the T-cell antigen receptor beta-chain rearrangements and respective mRNAs expressed. beta-chain cDNA clones were isolated from a library prepared from mRNA of aged killer T cells. The sequence of the beta-chain variable region element (VAK) was found to be identical with germ-line DNA. Four bases at the beta-chain diversity-joining region (D beta--J beta) junction cannot be explained by known germ-line D beta and J beta elements. These results illustrate that in T-cell clones altered antigen specificity correlates with a switch in productive beta-chain rearrangements of the T-cell receptor. When tested for its expression under physiological conditions, significant levels of VAK mRNA were found in normal lymphocyte populations.

A population-kinetical approach to RNA formation and degradation in growing and in resting cells.

Labelled RNA was extracted from growing and stationary cultures of the ciliate Tetrahymena and was separated chromatographically into poly(A)- and no poly(A)-containing fractions. A new method was used to derive from the data (cpm/A260, and tD, doubling time of RNA) absolute values of three growth terms which fully describe the population kinetics of RNA molecules: the rates of transcription, decay, and net growth. At all times the messenger RNA (mRNA) content of Tetrahymena was the result of a self-regulating equilibrium between synthesis and decay. The rates of transcription and of degradation of mRNA and ribosomal RNA (rRNA) were found to be controlled independently, but decay was dominant in establishing the growth-specific quantities per cell. In the stationary phase about 94% of all poly(A)-RNA molecules and about 50% of all mRNA molecules were kinetically silent. The remaining portions were transcribed with high rates, but also degraded immediately. During the culture growth cycle the rate of rRNA net growth responded positively to the cellular rRNA content suggesting an autocatalytic effect of rRNA on the rate of its accumulation.

Effects of benzo[a]pyrene diol epoxide adducts on DNA synthesis in mammalian cells.

The replication of DNA containing anti-benzo[a]pyrene diol epoxide (BPDE) adducts was studied in mammalian cells by first treating SV40 virus with BPDE in vitro, then infecting cells with virus containing a known number of adducts in the DNA. Viral transcription products necessary for replication were supplied by co-infection with an untreated virus containing a deletion as a DNA marker. Thus, only replicative effects of BPDE adducts were manifested. Delayed replication of the DNA from BPDE-treated virus, relative to the DNA containing the deletion, was observed, but in time most or all of the infecting molecules were able to replicate. The results are consistent with the hypothesis that adducts of BPDE in DNA block DNA synthesis in vivo, as they do in vitro, and that the block is gradually overcome by a repair mechanism that eliminates the adducts responsible for blockage or by delayed replicative bypass of the adducts. In spite of the ability of the system to overcome the delay in replication, the viability of the BPDE-treated virus in plaque assay was low, suggesting a persistent defect in transcription or a high level of error in repair or bypass replication.

Prereplicative events involving simian virus 40 DNA in permissive cells.

Simian virus 40 DNA molecules were found to be unable to replicate for 9 h after infection, even in cells that were already replicating the DNA of preinfecting simian virus 40; after 9 h, the ability of the DNA to replicate began to rise sharply. The kinetics of activation indicated that each DNA molecule undergoes a series of slow consecutive reactions, not involving T-antigen, before it can replicate. These pre-replicative molecular transformations probably involve configurational changes; their nature and their relation to the initiation of viral DNA synthesis is discussed. Observation of the replicative behavior of one viral DNA in the presence of another was made possible by the use of two different mutants with distinguishable DNAs: a viable deletion mutant containing DNA insensitive to TAqI restriction enzyme was used to provide viral functions required for replication, and a tsA mutant with TaqI-sensitive DNA was introduced at various times as a probe to determine the ability of the DNA to replicate under different conditions.

Changes in amount and synthesis of poly(A)- and poly(A)+ RNA in growing and resting cultures of Tetrahymena.

This investigation deals both qualitatively and quantitatively with the changes of RNA content and synthesis during the culture growth cycle of Tetrahymena. Affinity chromatography with an oligo(dT) column was used to separate poly(A)+ RNA from total RNA. The rates of synthesis of poly(A)- and poly(A)+ RNA were determined in terms of the incorporation of [5-3H]uridine. During the log phase, the cellular RNA and protein contents decreased steadily, whereas during the resting stage, both were constant. The extents of decrease of both fractions of RNA were essentially the same (54.4% and 50.6% for total and poly(A)+ RNA, respectively). Therefore, the relative contents of poly(A)+ RNA was constant from the beginning of the log to the resting stage (4.58%). The decrease in protein content, however, amounted to only 24.8%. Theoretically, a change in the age distribution during culture growth would cause a lower content of both fractions of RNA. The extents of the decrease in the rate of synthesis of both fractions were the same (75% and 79% for poly(A)- and poly(A)+ RNA, respectively). However, this reduction is so large that it cannot be solely the result of a shift of the age distribution of the cell population.