Differential gene expression in multistep tumorigenesis using an {\it in vitro\/} human cell model

Using a human terato-carcinoma cell line, PA-1, the functional role of the oncogenes and tumor suppressor gene involved in the multistep process of carcinogenesis have been analyzed. The expression of AP-2 was strongly correlated with the susceptibility to ras transformation. The differential responsiveness to growth factors between stage 1 ras resistant cells and stage 2 ras susceptible cells was observed, indicating that the ability of stage 2 cells to respond to the mutated ras oncogenes in transformation correlated with the ability to be stimulated by certain growth factors. Using differential screening of cDNA libraries, a number of differentially expressed cDNA clones was isolated. One of those, clone 12, is overexpressed in ras transformed stage 3 cells. The amino acid sequence of clone 12 is almost identical to a mouse LLrep3 gene that was growth-regulated, and 78% similar to a yeast ribosomal protein S4. These results suggest that the S4 gene may be involved in regulation of growth. Clone 9 is expressed in stage 1 ras resistant cells (3.5-kb and 3.0-kb transcripts) but the expression of this clone in stage 2 ras susceptible cells and stage 3 ras-transformed cells is greatly diminished. The expression of this cDNA clone was increased to at least five fold in ras resistant cells and nontumorigenic hybrids treated with retinoic acid but not increased in retinoic acid treated ras susceptible cells, ras transformed cells and the tumorigenic segregants. Partial sequence of this clone showed no homology to the sequences in Genbank. These findings suggest that clone 9 could be a suppressor gene or the genes that are involved in the biochemical pathway of tumor suppression or neurogenic differentiation. The apparent pleiotropic effect of the loss of this suppressor gene function support Harris' proposal that tumor suppressor genes regulate differentiation. The tumor suppressor gene may act as negative regulator of tumor growth by controlling gene expression in differentiation. ^

H2B histone gene expression during {\it Xenopus laevis\/} development

Histone gene expression is replication-independent during oogenesis and early embryogenesis in amphibians; however, it becomes replication-dependent during later embryogenesis and remains replication-dependent through adulthood. In order to understand the mechanism for this switch in transcriptional regulation of histone gene expression during amphibian development, linker-scanning mutations were made in a Xenopus laevis H2B histone gene promoter by oligonucleotide site-directed mutagenesis and assayed by microinjection into oocytes and embryos. The Xenopus H2B gene has a relatively simple promoter containing several transcriptional regulatory elements, including TFIID, CCAAT, and ATF motifs, required for maximal transcription in both oocytes and embryos. Factors binding to the CCAAT and ATF motifs are present in oocytes and embryos and increase slightly in abundance during early development. A sequence (CTTTACAT) in the frog H2B promoter resembling the conserved octamer motif (ATTTGCAT), the target for cell-cycle regulation of a human H2B gene, is additionally required for maximal H2B transcription in frog embryos. Oocytes and embryos contain multiple octamer-binding proteins that are expressed in a sequential manner during early development. Sequences encoding three novel octamer-binding proteins were isolated from Xenopus cDNA libraries by virtue of their similarity with the DNA binding (POU) domain of the ubiquitously expressed transcription factor Oct-1. The protein encoded by one of these genes, termed Oct-60, was localized mainly in the cytoplasm of oocytes and was also present in early embryos until the gastrula stage of development. Proteins encoded by the other two genes, Oct-25 and Oct-91, were present in embryos after the mid-blastula stage of development and decreased by early neurula stage. The activity of the Xenopus H2B octamer motif in embryos is not specifically associated with increased binding by Oct-1 or the appearance of novel octamer-binding proteins but requires the presence of an intact CCAAT motif. We found that synergistic interactions among promoter elements are important for full H2B promoter activity. The results suggest that transcription of the Xenopus H2B gene is replication-dependent when it is activated at the mid-blastula stage of development and that replication-dependent H2B transcription is mediated by Oct-1. ^

Molecular studies of human high grade B cell non -Hodgkin's lymphomas

The non-Hodgkin's B cell lymphomas are a diverse group of neoplastic diseases. The incidence rate of the malignant tumors has been rising rapidly over the past twenty years in the United States and worldwide. The lack of insight to pathogenesis of the disease poses a significant problem in the early detection and effective treatment of the human malignancies. These studies attempted to investigate the molecular basis of pathogenesis of the human high grade B cell non-Hodgkin's lymphomas with a reverse genetic approach. The specific objective was to clone gene(s) which may play roles in development and progression of human high grade B cell non-Hodgkin's lymphomas.^ The messenger RNAs from two high grade B cell lymphoma lines, CJ and RR, were used for construction of cDNA libraries. Differential screening of the derived cDNA libraries yielded a 1.4 kb cDNA clone. The gene, designated as NHL-B1.4, was shown to be highly amplified and over-expressed in the high grade B cell lymphoma lines. It was not expressed in the peripheral blood lymphoid cells from normal donors. However, it was inducible in peripheral blood T lymphocytes by a T cell mitogen, PHA, but could not be activated in normal B cells by B cell mitogen PMA. Further molecular characterization revealed that the gene may have been rearranged in the RR and some other B cell lymphoma lines. The coding capacity of the cDNA has been confirmed by a rabbit reticulocyte lysate and wheat germ protein synthesis system. A recombinant protein with a molecular weight of approximate 30 kDa was visualized in autoradiogram. Polyclonal antisera have been generated by immunization of two rabbits with the NHL-B1.4 recombinant protein produced in the E. coli JM109. The derived antibody can recognize a natural protein with molecular weight of 49 kDa in cell lysate of activated peripheral T lymphocytes of normal donors and both the cell lysate and supernatant of RR B cell lymphoma lines. The possible biologic functions of the molecule has been tested preliminarily in a B lymphocyte proliferation assay. It was found that the Q-sepharose chromatograph purified supernatant of COS cell transfection could increase tritiated thymidine uptake by B lymphocytes but not by T lymphocytes. The B cell stimulatory activity of the supernatant of COS cell tranfection could be neutralized by the polyclonal antisera, indicating that the NHL-B1.4 gene product may be a molecule with BCGF-like activity.^ The expression profiles of NHL-B1.4 in normal and neoplastic lymphoid cells were consistent with the current B lymphocyte activation model and autocrine hypothesis of high grade B cell lymphomagenesis. These results suggested that the NHL-B1.4 cDNA may be a disease-related gene of human high grade B cell lymphomas, which may codes for a postulated B cell autocrine growth factor. ^

Biosynthetic origin of conjugated double bonds: Production of fatty acid components of high-value drying oils in transgenic soybean embryos

Vegetable oils that contain fatty acids with conjugated double bonds, such as tung oil, are valuable drying agents in paints, varnishes, and inks. Although several reaction mechanisms have been proposed, little is known of the biosynthetic origin of conjugated double bonds in plant fatty acids. An expressed sequence tag (EST) approach was undertaken to characterize the enzymatic basis for the formation of the conjugated double bonds of α-eleostearic (18:3Δ9cis,11trans,13trans) and α-parinaric (18:4Δ9cis,11trans,13trans,15cis) acids. Approximately 3,000 ESTs were generated from cDNA libraries prepared from developing seeds of Momordica charantia and Impatiens balsamina, tissues that accumulate large amounts of α-eleostearic and α-parinaric acids, respectively. From ESTs of both species, a class of cDNAs encoding a diverged form of the Δ12-oleic acid desaturase was identified. Expression of full-length cDNAs for the Momordica (MomoFadX) and Impatiens (ImpFadX) enzymes in somatic soybean embryos resulted in the accumulation of α-eleostearic and α-parinaric acids, neither of which is present in untransformed soybean embryos. α-Eleostearic and α-parinaric acids together accounted for as much as 17% (wt/wt) of the total fatty acids of embryos expressing MomoFadX. These results demonstrate the ability to produce fatty acid components of high-value drying oils in transgenic plants. These findings also demonstrate a previously uncharacterized activity for Δ12-oleic acid desaturase-type enzymes that we have termed “conjugase.”

Gene discovery in the wood-forming tissues of poplar: Analysis of 5,692 expressed sequence tags

A rapidly growing area of genome research is the generation of expressed sequence tags (ESTs) in which large numbers of randomly selected cDNA clones are partially sequenced. The collection of ESTs reflects the level and complexity of gene expression in the sampled tissue. To date, the majority of plant ESTs are from nonwoody plants such as Arabidopsis, Brassica, maize, and rice. Here, we present a large-scale production of ESTs from the wood-forming tissues of two poplars, Populus tremula L. × tremuloides Michx. and Populus trichocarpa ‘Trichobel.’ The 5,692 ESTs analyzed represented a total of 3,719 unique transcripts for the two cDNA libraries. Putative functions could be assigned to 2,245 of these transcripts that corresponded to 820 protein functions. Of specific interest to forest biotechnology are the 4% of ESTs involved in various processes of cell wall formation, such as lignin and cellulose synthesis, 5% similar to developmental regulators and members of known signal transduction pathways, and 2% involved in hormone biosynthesis. An additional 12% of the ESTs showed no significant similarity to any other DNA or protein sequences in existing databases. The absence of these sequences from public databases may indicate a specific role for these proteins in wood formation. The cDNA libraries and the accompanying database are valuable resources for forest research directed toward understanding the genetic control of wood formation and future endeavors to modify wood and fiber properties for industrial use.

Circadian clock-controlled genes isolated from Neurospora crassa are late night- to early morning-specific

An endogenous circadian biological clock controls the temporal aspects of life in most organisms, including rhythmic control of genes involved in clock output pathways. In the fungus Neurospora crassa, one pathway known to be under control of the clock is asexual spore (conidia) development. To understand more fully the processes that are regulated by the N. crassa circadian clock, systematic screens were carried out for genes that oscillate at the transcriptional level. Time-of-day-specific cDNA libraries were generated and used in differential screens to identify six new clock-controlled genes (ccgs). Transcripts specific for each of the ccgs preferentially accumulate during the late night to early morning, although they vary with respect to steady-state mRNA levels and amplitude of the rhythm. Sequencing of the ends of the new ccg cDNAs revealed that ccg-12 is identical to N. crassa cmt encoding copper metallothionein, providing the suggestion that not all clock-regulated genes in N. crassa are specifically involved in the development of conidia. This was supported by finding that half of the new ccgs, including cmt(ccg-12), are not transcriptionally induced by developmental or light signals. These data suggest a major role for the clock in the regulation of biological processes distinct from development.

The diversity and evolutionary relationships of the pregnancy-associated glycoproteins, an aspartic proteinase subfamily consisting of many trophoblast-expressed genes

The pregnancy-associated glycoproteins (PAGs) are structurally related to the pepsins, thought to be restricted to the hooved (ungulate) mammals and characterized by being expressed specifically in the outer epithelial cell layer (chorion/trophectoderm) of the placenta. At least some PAGs are catalytically inactive as proteinases, although each appears to possess a cleft capable of binding peptides. By cloning expressed genes from ovine and bovine placental cDNA libraries, by Southern genomic blotting, by screening genomic libraries, and by using PCR to amplify portions of PAG genes from genomic DNA, we estimate that cattle, sheep, and most probably all ruminant Artiodactyla possess many, possibly 100 or more, PAG genes, many of which are placentally expressed. The PAGs are highly diverse in sequence, with regions of hypervariability confined largely to surface-exposed loops. Nonsynonymous (replacement) mutations in the regions of the genes coding for these hypervariable loop segments have accumulated at a higher rate than synonymous (silent) mutations. Construction of distance phylograms, based on comparisons of PAG and related aspartic proteinase amino acid sequences, suggests that much diversification of the PAG genes occurred after the divergence of the Artiodactyla and Perissodactyla, but that at least one gene is represented outside the hooved species. The results also suggest that positive selection of duplicated genes has acted to provide considerable functional diversity among the PAGs, whose presence at the interface between the placenta and endometrium and in the maternal circulation indicates involvement in fetal–maternal interactions.

Reciprocal subtraction differential RNA display: An efficient and rapid procedure for isolating differentially expressed gene sequences

A reciprocal subtraction differential RNA display (RSDD) approach has been developed that permits the rapid and efficient identification and cloning of both abundant and rare differentially expressed genes. RSDD comprises reciprocal subtraction of cDNA libraries followed by differential RNA display. The RSDD strategy was applied to analyze the gene expression alterations resulting during cancer progression as adenovirus-transformed rodent cells developed an aggressive transformed state, as documented by elevated anchorage-independence and enhanced in vivo oncogenesis in nude mice. This approach resulted in the identification and cloning of both known and a high proportion (>65%) of unknown sequences, including cDNAs displaying elevated expression as a function of progression (progression-elevated gene) and cDNAs displaying suppressed expression as a function of progression (progression-suppressed gene). Sixteen differentially expressed genes, including five unknown progression-elevated genes and six unknown progression-suppressed genes, have been characterized. The RSDD scheme should find wide application for the effective detection and isolation of differentially expressed genes.

A green fluorescent protein-reporter mammalian two-hybrid system with extrachromosomal maintenance of a prey expression plasmid: Application to interaction screening

An improved mammalian two-hybrid system designed for interaction trap screening is described in this paper. CV-1/EBNA-1 monkey kidney epithelial cells expressing Epstein–Barr virus nuclear antigen 1 (EBNA-1) were stably transfected with a reporter plasmid for GAL4-dependent expression of the green fluorescent protein (GFP). A resulting clone, GB133, expressed GFP strongly when transfected transiently with transcriptional activators fused to GAL4 DNA-binding domain with minimal background GFP expression. GB133 cells maintained plasmids containing the OriP Epstein–Barr virus replication origin that directs replication of plasmids in mammalian cells in the presence of the EBNA-1 protein. GB133 cells transfected stably with a model bait expressed GFP when further transfected transiently with an expression plasmid for a known positive prey. When the bait-expressing GB133 cells were transfected transiently with an OriP-containing expression plasmid for the positive prey together with excess amounts of empty vector, cells that received the positive prey were readily identified by green fluorescence in cell culture and eventually formed green fluorescent microcolonies, because the prey plasmid was maintained by the EBNA-1/Ori-P system. The green fluorescent microcolonies were harvested directly from the culture dishes under a fluorescence microscope, and total DNA was then prepared. Prey-encoding cDNA was recovered by PCR using primers annealing to the vector sequences flanking the insert-cloning site. This system should be useful in mammalian cells for efficient screening of cDNA libraries by two-hybrid interaction.

Leptin can induce proliferation, differentiation, and functional activation of hemopoietic cells

Many cytokines exert their biological effect through members of the hemopoietin receptor family. Using degenerate oligonucleotides to the common WSXWS motif, we have cloned from human hemopoietic cell cDNA libraries various forms of the receptor that was recently shown to bind the obesity hormone, leptin. mRNAs encoding long and short forms of the human leptin receptor were found to be coexpressed in a range of human and murine hemopoietic organs, and a subset of cells from these tissues bound leptin at the cell surface. Ectopic expression in murine Ba/F3 and M1 cell lines revealed that the long, but not the short, form of the leptin receptor can signal proliferation and differentiation, respectively. In cultures of murine or human marrow cells, human leptin exhibited no capacity to stimulate cell survival or proliferation, but it enhanced cytokine production and phagocytosis of Leishmania parasites by murine peritoneal macrophages. Our data provide evidence that, in addition to its role in fat regulation, leptin may also be able to regulate aspects of hemopoiesis and macrophage function.

Androgen-induced proliferative quiescence in prostate cancer cells: The role of AS3 as its mediator

In the prostate gland of adult mammals, most epithelial cells are in a state of proliferative quiescence. Androgens regulate this effect by inducing cell cycle arrest in the G0/G1 phase. Potential mediators of this androgen-induced proliferative shutoff were identified by means of subtracted cDNA libraries. The expression pattern of one of these sequences, AS3, strongly correlated with the expression of the androgen-induced proliferative shutoff both temporally and dosewise. The AS3 gene is located on chromosome 13 q12.3, in close proximity to the BRCA2 gene. The loss of chromosomal regions where AS3 alleles are located correlates with various human cancers, including prostate. The biological effect of AS3 was tested in two stable cell lines, one expressing sense and another expressing antisense AS3 constructs, both under tetracycline regulation. S9 cells were obtained by retroviral infection with virions containing a tetracycline-regulated sense AS3 construct. In these cells, sense AS3 was negatively regulated by tetracycline. Tetracycline withdrawal increased the expression of AS3 mRNA and protein. The expression of tetracycline-regulated AS3 resulted in inhibition of cell proliferation. A4 cells were obtained by retroviral infection with virions containing a tetracycline-regulated antisense AS3 construct. Vector-driven expression of antisense-AS3 blocked the induction of androgen-induced endogenous AS3 mRNA and blocked the inhibitory effect of androgens on cell proliferation. Tetracycline-regulated expression of the empty vector control had no effect on cell proliferation. These experiments strongly suggest that AS3 is a mediator of the androgen-induced proliferative shutoff.

PAGE-1, an X chromosome-linked GAGE-like gene that is expressed in normal and neoplastic prostate, testis, and uterus

We have used a combination of computerized database mining and experimental expression analyses to identify a gene that is preferentially expressed in normal male and female reproductive tissues, prostate, testis, fallopian tube, uterus, and placenta, as well as in prostate cancer, testicular cancer, and uterine cancer. This gene is located on the human X chromosome, and it is homologous to a family of genes encoding GAGE-like proteins. GAGE proteins are expressed in a variety of tumors and in testis. We designate the novel gene PAGE-1 because the expression pattern in the Cancer Genome Anatomy Project libraries indicates that it is predominantly expressed in normal and neoplastic prostate. Further database analysis indicates the presence of other genes with high homology to PAGE-1, which were found in cDNA libraries derived from testis, pooled libraries (with testis), and in a germ cell tumor library. The expression of PAGE-1 in normal and malignant prostate, testicular, and uterine tissues makes it a possible target for the diagnosis and possibly for the vaccine-based therapy of neoplasms of prostate, testis, and uterus.

A 104-kDa diacylglycerol kinase containing ankyrin-like repeats localizes in the cell nucleus.

The cDNA corresponding to a fourth species of diacylglycerol (DG) kinase (EC 2.7.1.107) was isolated from cDNA libraries of rat retina and brain. This cDNA encoded a 929-aa, 104-kDa polypeptide termed DGK-IV. DGK-IV was different from previously identified mammalian DG kinase species, DGK-I, DGK-II, and DGK-III, in that it contained no EF-hand motifs but did contain four ankyrin-like repeats at the carboxyl terminus. These structural features of DGK-IV closely resemble the recently cloned, eye-specific DG kinase of Drosophila that is encoded by the retinal degeneration A (rdgA) gene. However, DGK-IV was expressed primarily in the thymus and brain with relatively low expression in the eye and intestine. Furthermore, the primary structure of the DGK-IV included a nuclear targeting motif, and immunocytochemical analysis revealed DGK-IV to localize in the nucleus of COS-7 cells transfected with the epitope-tagged cDNA, suggesting an involvement of DGK-IV in intranuclear processes.

Identification of novel genes in Drosophila reveals the complex regulation of early gene activity in the mesoderm.

Two zygotic genes, twist and snail, are indispensable for the correct establishment of the mesoderm primordium in the early Drosophila embryo. They are also needed for morphogenesis and differentiation of the mesoderm. Both genes code for transcription factors with different, albeit complementary, functions. Therefore, to understand the early development of the mesoderm, it will be necessary to identify and study the genes regulated by twist and snail. We have searched for downstream genes using a subtractive cDNA library enriched in sequences expressed in the mesoderm. We have isolated sequences that correspond to 13 novel early mesoderm genes. These novel genes show a variety of expression patterns and also differ in their dependence on twist and snail functions. This indicates that the regulation of early gene activity in the mesoderm is more complex than previously thought.

Cloning and characterization of cDNAs for matrix metalloproteinases of regenerating newt limbs.

Matrix metalloproteinases (MMPs) of regenerating urodele limbs have been suggested to play crucial roles in the process of the dedifferentiation of cells in the damaged tissues and the ensuing blastema formation because the activation of MMPs is an early and conspicuous event occurring in the amputated limb. MMP cDNAs were cloned as products of the reverse transcription-PCR from cDNA libraries of newt limbs, and their structures were characterized. Three cDNAs encoding newt MMPs (2D-1, 2D-19, and 2D-24) have been cloned from second day postamputation regenerating limbs, and a cDNA (EB-1) was cloned from early bud-stage regenerating limbs. These cDNAs included the full-length coding regions. The deduced amino acid sequences of 2D-1, 2D-19, 2D-24, and EB-1 had a homology with mammalian MMP9, MMP3/10, MMP3/10, and MMP13, respectively. The basic motif of these newt MMP genes was similar to mammalian counterparts and contained regions encoding a putative signal sequence, a propeptide, an active site with three zinc-binding histidine residues, a calcium-binding domain, a hemopexin region, and three key cysteine residues. However, some unique molecular evolutionary features were also found in the newt MMPs. cDNAs of 2D-19 and 2D-24 contained a specific insertion and deletion, respectively. The insertion of 2D-19 is threonine-rich, similar to the threonine cluster found in the collagenase-like sea urchin hatching enzyme. Northern blot analysis showed that the expression levels of the newt MMPs were dramatically increased after amputation, suggesting that they play an important role(s) in tissue remodeling of the regenerating limb.