Functional properties of non-muscle tropomyosin isoforms.

Tropomyosins are a family of actin filament binding proteins. They have been identified in many organisms, including yeast, nematodes, Drosophila, birds and mammals. In metazoans, different forms of tropomyosin are characteristic of specific cell types. Most non-muscle cells, such as fibroblasts, express five to eight isoforms of tropomyosins. The various isoforms exhibit distinct biochemical properties that appear to be required for specific cellular functions.

In vitro and in vivo characterization of four fibroblast tropomyosins produced in bacteria: TM-2, TM-3, TM-5a, and TM-5b are co-localized in interphase fibroblasts.

Most cell types express several tropomyosin isoforms, the individual functions of which are poorly understood. In rat fibroblasts there are at least six isoforms; TM-1, TM-2, TM-3, TM-4, TM-5a, and TM-5b. TM-1 is the product of the beta gene. TM-4 is produced from the TM-4 gene, and TMs 2, 3, 5a, and 5b are the products of the alpha gene. To begin to study the localization and function of the isoforms in fibroblasts, cDNAs for TM isoforms 2, 3, 5a, and 5b were placed into bacterial expression vectors and used to produce TM isoforms. The bacterially produced TMs were determined to be full length by sequencing the amino- and carboxy termini. These TMs were found to bind to F-actin in vitro, with properties similar to that of skeletal muscle TM. In addition, competition experiments demonstrated that TM-5b was better than TM-5a in displacing other TM isoforms from F-actin in vitro. To investigate the intracellular localization of these fibroblast isoforms, each was derivatized with a fluorescent chromophore and microinjected into rat fibroblasts. TM-2, TM-3, TM-5a, and TM-5b were each found to associate along actin filaments. There was no preferred cellular location or subset of actin filaments for these isoforms. Furthermore, co-injection of two isoforms labeled with different fluorochromes showed identical staining. At the level of the light microscope, these isoforms from the alpha gene do not appear to achieve different functions by binding to particular subsets of actin filaments or locations in cells. Some alternative possibilities are discussed. The results show that bacterially produced TMs can be used to study in vitro and in vivo properties of the isoforms.

Tropomyosin isoforms in chicken embryo fibroblasts: purification, characterization, and changes in Rous sarcoma virus-transformed cells.

Seven polypeptides (a, b, c, 1, 2, 3a, and 3b) have been previously identified as tropomyosin isoforms in chicken embryo fibroblasts (CEF) (Lin, J. J.-C., Matsumura, F., and Yamashiro-Matsumura, S., 1984, J. Cell. Biol., 98:116-127). Spots a and c had identical mobility on two-dimensional gels with the slow-migrating and fast-migrating components, respectively, of chicken gizzard tropomyosin. However, the remaining isoforms of CEF tropomyosin were distinct from chicken skeletal and cardiac tropomyosins on two-dimensional gels. The mixture of CEF tropomyosin has been isolated by the combination of Triton/glycerol extraction of monolayer cells, heat treatment, and ammonium sulfate fractionation. The yield of tropomyosin was estimated to be 1.4% of total CEF proteins. The identical set of tropomyosin isoforms could be found in the antitropomyosin immunoprecipitates after the cell-free translation products of total poly(A)+ RNAs isolated from CEF cells. This suggested that at least seven mRNAs coding for these tropomyosin isoforms existed in the cell. Purified tropomyosins (particularly 1, 2, and 3) showed different actin-binding abilities in the presence of 100 mM KCl and no divalent cation. Under this condition, the binding of tropomyosin 3 (3a + 3b) to actin filaments was significantly weaker than that of tropomyosin 1 or 2. CEF tropomyosin 1, and probably 3, could be cross-linked to form homodimers by treatment with 5,5'-dithiobis-(2-nitrobenzoate), whereas tropomyosin a and c formed a heterodimer. These dimer species may reflect the in vivo assembly of tropomyosin isoforms, since dimer formation occurred not only with purified tropomyosin but also with microfilament-associated tropomyosin. The expression of these tropomyosin isoforms in Rous sarcoma virus-transformed CEF cells has also been investigated. In agreement with the previous report by Hendricks and Weintraub (Proc. Natl. Acad. Sci. USA., 78:5633-5637), we found that major tropomyosin 1 was greatly reduced in transformed cells. We have also found that the relative amounts of tropomyosin 3a and 3b were increased in both the total cell lysate and the microfilament fraction of transformed cells. Because of the different actin-binding properties observed for CEF tropomyosins, changes in the expression of these isoforms may, in part, be responsible for the reduction of actin cables and the alteration of cell shape found in transformed cells.

Regulation of alternative splicing in vivo by overexpression of antagonistic splicing factors.

The opposing effects of SF2/ASF and heterogeneous nuclear ribonucleoprotein (hnRNP) A1 influence alternative splicing in vitro. SF2/ASF or hnRNP A1 complementary DNAs were transiently overexpressed in HeLa cells, and the effect on alternative splicing of several cotransfected reporter genes was measured. Increased expression of SF2/ASF activated proximal 5' splice sites, promoted inclusion of a neuron-specific exon, and prevented abnormal exon skipping. Increased expression of hnRNP A1 activated distal 5' splice sites. Therefore, variations in the intracellular levels of antagonistic splicing factors influence different modes of alternative splicing in vivo and may be a natural mechanism for tissue-specific or developmental regulation of gene expression.

Cytoskeletal changes in cell transformation and tumorigenesis.

Research during the past couple of years has provided important new information as to how the actin cytoskeleton contributes to growth control in both normal and transformed cells. The cytoskeleton can no longer be viewed as simply a structural framework playing a role in cell shape and motile events such as cell movement, intracellular transport, contractile-ring formation and chromosome movement. More recent experiments show that the cytoskeleton plays a critical role in the regulation of various cellular processes linked to transformation including proliferation, contact inhibition, anchorage-independent cell growth, and apoptosis.

Modulation of exon skipping and inclusion by heterogeneous nuclear ribonucleoprotein A1 and pre-mRNA splicing factor SF2/ASF.

The essential splicing factor SF2/ASF and the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) modulate alternative splicing in vitro of pre-mRNAs that contain 5' splice sites of comparable strengths competing for a common 3' splice site. Using natural and model pre-mRNAs, we have examined whether the ratio of SF2/ASF to hnRNP A1 also regulates other modes of alternative splicing in vitro. We found that an excess of SF2/ASF effectively prevents inappropriate exon skipping and also influences the selection of mutually exclusive tissue-specific exons in natural beta-tropomyosin pre-mRNA. In contrast, an excess of hnRNP A1 does not cause inappropriate exon skipping in natural constitutively or alternatively spliced pre-mRNAs. Although hnRNP A1 can promote alternative exon skipping, this effect is not universal and is dependent, e.g., on the size of the internal alternative exon and on the strength of the polypyrimidine tract in the preceding intron. With appropriate alternative exons, an excess of SF2/ASF promotes exon inclusion, whereas an excess of hnRNP A1 causes exon skipping. We propose that in some cases the ratio of SF2/ASF to hnRNP A1 may play a role in regulating alternative splicing by exon inclusion or skipping through the antagonistic effects of these proteins on alternative splice site selection.

Nonmuscle and muscle tropomyosin isoforms are expressed from a single gene by alternative RNA splicing and polyadenylation.

The molecular basis for the expression of rat embryonic fibroblast tropomyosin 1 and skeletal muscle beta-tropomyosin was determined. cDNA clones encoding these tropomyosin isoforms exhibit complete identity except for two carboxy-proximal regions (amino acids 189 to 213 and 258 to 284) and different 3'-untranslated sequences. The isoform-specific regions delineate the troponin T-binding domains of skeletal muscle tropomyosin. Analysis of genomic clones indicates that there are two separate loci in the rat genome that contain sequences complementary to these mRNAs. One locus is a pseudogene. The other locus contains a single gene made up of 11 exons and spans approximately 10 kilobases. Sequences common to all mRNAs were found in exons 1 through 5 (amino acids 1 to 188) and exons 8 and 9 (amino acids 214 to 257). Exons 6 and 11 are specific for fibroblast mRNA (amino acids 189 to 213 and 258 to 284, respectively), while exons 7 and 10 are specific for skeletal muscle mRNA (amino acids 189 to 213 and 258 to 284, respectively). In addition, exons 10 and 11 each contain the entire 3'-untranslated sequences of the respective mRNAs including the polyadenylation site. Although the gene is also expressed in smooth muscle (stomach, uterus, and vas deferens), only the fibroblast-type splice products can be detected in these tissues. S1 and primer extension analyses indicate that all mRNAs expressed from this gene are transcribed from a single promoter. The promoter was found to contain G-C-rich sequences, a TATA-like sequence TTTTA, no identifiable CCAAT box, and two putative Sp1-binding sites.

Polypyrimidine tract-binding protein positively regulates inclusion of an alternative 3'-terminal exon.

Polypyrimidine tract-binding protein (PTB) is an abundant vertebrate hnRNP protein. PTB binding sites have been found within introns both upstream and downstream of alternative exons in a number of genes that are negatively controlled by the binding of PTB. We have previously reported that PTB binds to a pyrimidine tract within an RNA processing enhancer located adjacent to an alternative 3'-terminal exon within the gene coding for calcitonin and calcitonin gene-related peptide. The enhancer consists of a pyrimidine tract and CAG directly abutting on a 5' splice site sequence to form a pseudoexon. Here we show that the binding of PTB to the enhancer pyrimidine tract is functional in that exon inclusion increases when in vivo levels of PTB increase. This is the first example of positive regulation of exon inclusion by PTB. The binding of PTB was antagonistic to the binding of U2AF to the enhancer-located pyrimidine tract. Altering the enhancer pyrimidine tract to a consensus sequence for the binding of U2AF eliminated enhancement of exon inclusion in vivo and exon polyadenylation in vitro. An additional PTB binding site was identified close to the AAUAAA hexanucleotide sequence of the exon 4 poly(A) site. These observations suggest a dual role for PTB in facilitating recognition of exon 4: binding to the enhancer pyrimidine tract to interrupt productive recognition of the enhancer pseudoexon by splicing factors and interacting with the poly(A) site to positively affect polyadenylation.

Molecular cloning and in vitro expression of a cDNA clone for human cellular tumor antigen p53.

Three clones for the human tumor antigen p53 were isolated from a cDNA library prepared from A431 cells. One of these clones, pR4-2, contains the entire coding region for human p53. This clone directs the synthesis of a polypeptide with the correct molecular weight and immunological epitopes of an authentic p53 molecule in an in vitro transcription-translation reaction. Although the pR4-2 clone contains the coding region for p53, it is not a full-length copy of the human p53 mRNA. Northern analysis showed that the p53 mRNA is approximately 2,500 nucleotides long, whereas the pR4-2 insert is only 1,760 base pairs in length. Analysis of the DNA sequence of this clone suggests that the human p53 polypeptide has 393 amino acids. We compared the predicted amino acid sequence of the pR4-2 clone with similar clones for the mouse p53 and found long regions of amino acid homology between these two molecules.

Three novel brain tropomyosin isoforms are expressed from the rat alpha-tropomyosin gene through the use of alternative promoters and alternative RNA processing.

cDNA clones encoding three novel tropomyosins, termed TMBr-1, TMBr-2, and TMBr-3, were isolated and characterized from a rat brain cDNA library. All are derived from a single gene, which was previously found to express striated muscle alpha-tropomyosin and a number of other tropomyosin isoforms via an alternative splicing mechanism (N. Ruiz-Opazo and B. Nadal-Ginard, J. Biol. Chem. 262:4755-4765, 1987; D. F. Wieczorek, C. W. J. Smith, and B. Nadal-Ginard, Mol. Cell. Biol. 8:679-694, 1988). The derived amino acid sequences revealed that TMBr-1 contains 281 amino acids, TMBr-2 contains 251 amino acids, and TMBr-3 contains 245 amino acids. All three proteins contain a region that is identical to amino acids 81 through 258 of skeletal muscle alpha-tropomyosin. TMBr-1 is identical to striated muscle alpha-tropomyosin from amino acids 1 through 258 but contains a novel COOH-terminal region from amino acids 259 through 281. TMBr-2 and TMBr-3 both contain identical NH2-terminal sequences from amino acids 1 through 44 which were found to be expressed from a novel promoter. TMBr-3 contains the same COOH-terminal region as TMBr-1, whereas TMBr-2 contains a second novel COOH-terminal region. The genomic organization of the exons encoding TMBr-1, TMBr-2, and TMBr-3 were determined. These studies revealed a previously uncharacterized promoter located in the internal region of the alpha-TM gene as well as two novel COOH-terminal coding exons. The alpha-TM gene is a complex transcription unit containing 15 exons including two alternative promoters, two internal mutually exclusive exon cassettes, and four alternatively spliced 3' exons that encode four different COOH-terminal coding regions. A total of nine distinct mRNAs are known to be expressed from the alpha-TM gene in a cell type-specific manner in tissues such as striated muscle, smooth muscle, kidney, liver, brain, and fibroblasts. The mRNAs encoding TMBr-1, TMBr-2, and TMBr-3 were found to be expressed only in brain tissue, with TMBr-3 being expressed at much greater levels than TMBr-1 and TMBr-2. The individual structural characteristics of each brain alpha-tropomyosin isoform and their possible functions are discussed.

Caldesmon inhibits nonmuscle cell contractility and interferes with the formation of focal adhesions.

Caldesmon is known to inhibit the ATPase activity of actomyosin in a Ca(2+)-calmodulin-regulated manner. Although a nonmuscle isoform of caldesmon is widely expressed, its functional role has not yet been elucidated. We studied the effects of nonmuscle caldesmon on cellular contractility, actin cytoskeletal organization, and the formation of focal adhesions in fibroblasts. Transient transfection of nonmuscle caldesmon prevents myosin II-dependent cell contractility and induces a decrease in the number and size of tyrosine-phosphorylated focal adhesions. Expression of caldesmon interferes with Rho A-V14-mediated formation of focal adhesions and stress fibers as well as with formation of focal adhesions induced by microtubule disruption. This inhibitory effect depends on the actin- and myosin-binding regions of caldesmon, because a truncated variant lacking both of these regions is inactive. The effects of caldesmon are blocked by the ionophore A23187, thapsigargin, and membrane depolarization, presumably because of the ability of Ca(2+)-calmodulin or Ca(2+)-S100 proteins to antagonize the inhibitory function of caldesmon on actomyosin contraction. These results indicate a role for nonmuscle caldesmon in the physiological regulation of actomyosin contractility and adhesion-dependent signaling and further demonstrate the involvement of contractility in focal adhesion formation.

Phospholipid-sensitive Ca2+-dependent protein phosphorylation system in various types of leukemic cells from human patients and in human leukemic cell lines HL60 and K562, and its inhibition by alkyl-lysophospholipid.

Phospholipid-sensitive Ca2+-dependent protein kinase (PL-Ca-PK), its endogenous substrate proteins, and regulation of the enzyme system by an antitumor agent alkyl-lysophospholipid were investigated in various types of leukemic cells (chronic myelocytic, acute myelocytic, and acute monocytic) from patients and in two cultured human leukemic cell lines (HL60 and K562). Exceedingly high levels of PL-Ca-PK, largely localized in the particulate fraction, were found in all types and lines of leukemic cells; much lower levels of cyclic adenosine 3':5'-monophosphate-dependent protein kinase and cyclic guanosine 3':5'-monophosphate-dependent protein kinase were also found. Although numerous and similar endogenous substrates for PL-Ca-PK were found in all cell types and lines examined, substrates specific for certain leukemic cells appeared to be present. Substrate proteins for calmodulin-sensitive Ca2+-dependent protein kinase, cyclic adenosine 3':5'-monophosphate-dependent protein kinase, and cyclic guanosine 3':5'-monophosphate-dependent protein kinase, in comparison, were much fewer or undetected. The PL-Ca-PK activity and the phosphatidylserine-Ca2+-stimulated phosphorylation of endogenous proteins from leukemic cells were inhibited by alkyl-lysophospholipid, which acted as a competitive inhibitor of the phospholipid cofactor of the enzyme. The findings suggested that the PL-Ca-PK system is a predominant protein phosphorylation system in leukemic cells and that this enzyme system may represent a site of cytotoxic action of alkyl-lysophospholipid.

Loss of MLCK leads to disruption of cell-cell adhesion and invasive behavior of breast epithelial cells via increased expression of EGFR and ERK/JNK signaling.

Myosin light chain kinase (MLCK) expression is downregulated in breast cancer, including invasive ductal carcinoma compared with ductal breast carcinoma in situ and metastatic breast tumors. However, little is known about how loss of MLCK expression contributes to tumor progression. MLCK is a component of the actin cytoskeleton and its known role is the phosphorylation of the regulatory light chain of myosin II. To gain insights into the role of MLCK in breast cancer, we perturbed its function using small interfering RNA (siRNA) or pharmacological inhibition in untransformed breast epithelial cells (MCF10A). Loss of MLCK by siRNAs led to increased cell migration and invasion, disruption of cell-cell adhesions and enhanced formation of focal adhesions at the leading edge of migratory cells. In addition, downregulation of MLCK cooperated with HER2 in MCF10A cells to promote cell migration and invasion and low levels of MLCK is associated with a poor prognosis in HER2-positive breast cancer patients. Associated with these altered migratory behaviors were increased expression of epidermal growth factor receptor and activation of extracellular signal-regulated kinase and c-Jun N-terminal kinase signaling pathways in MLCK downregulated MCF10A cells. By contrast, inhibition of the kinase function of MLCK using pharmacological agents inhibited cell migration and invasion, and did not affect cellular adhesions. Our results show that loss of MLCK contributes to the migratory properties of epithelial cells resulting from changes in cell-cell and cell-matrix adhesions, and increased epidermal growth factor receptor signaling. These findings suggest that decreased expression of MLCK may have a critical role during tumor progression by facilitating the metastatic potential of tumor cells.

Differential regulation of calcium-dependent and calcium-independent cyclic nucleotide phosphodiesterases from heart by palmitoylcarnitine and palmitoyl coenzyme A.

Regulation of Ca2+-dependent (peak I) and Ca2+-independent (peak II) phosphodiesterases from the heart by various fatty acyl esters and phospholipids were studied. DL-Palmitoylcarnitine stimulated the basal activity (in the absence of Ca2+) of peak I enzyme, while non-competitively inhibiting peak II enzyme with respect to cyclic AMP. It had no effect on other species of Ca2+-independent phosphodiesterases, including cyclic AMP- and cyclic GMP-specific enzymes from the lung, and cyclic CMP enzyme from the liver Palmitoyl-CoA and phosphatidylserine also stimulated the basal activity of peak I enzyme, but they were without effect on peak II enzyme. In comparison, DL-palmitoylcarnitine inhibited Ca2+-dependent activity of cardiac myosin light chain kinase, whereas phosphatidylserine was without effect. It is conceivable that differential regulation of phosphodiesterases by these lipids could profoundly alter the levels or effects, or both, of cyclic nucleotides and Ca2+ in the myocardium.

Expression of smooth muscle and nonmuscle tropomyosins in Escherichia coli and characterization of bacterially produced tropomyosins.

The cDNA encoding the beta-tropomyosin isoform of chicken smooth muscle (CSM beta) was constructed and expressed in Escherichia coli to produce recombinant, unacetylated beta-tropomyosin (rCSM beta) and a mutant (rCSM beta-7) with a 7-residue deletion at its amino-terminus. Furthermore, the cDNA coding for human fibroblast tropomyosin isoform 3 (hTM3) was also used to produce unacetylated hTM3 (called PEThTM3). All of bacterially-made tropomyosins were high alpha-helical in structure as judged by CD analysis and resistant to heat denaturation. Both the rCSM beta and PEThTM3 exhibited saturable binding to F-actin with apparent binding constants of 1.14 x 10(6) and 2.78 x 10(6) M-1, respectively. The bacterially made, unacetylated smooth muscle tropomyosin (rCSM beta) appeared to have a comparable actin-binding affinity to that of gel-purified CSM beta homodimer (1.25 x 10(6) M-1) but significantly lower than that for native gizzard tropomyosin (CSM-TM) heterodimer (1.28 x 10(7) M-1). The amino-terminal deletion mutant rCSM beta-7 failed to bind to F-actin. Effects of gizzard caldesmon on the actin binding of these bacterially made tropomyosins were also examined. Under the binding condition containing 0.5 mM MgCl2 and 30 mM KCl, caldesmon greatly enhanced the binding of rCSM beta to F-actin. However, under the same condition, there was a slight enhancement in the actin-binding for gel-purified CSM beta or PEThTM3 (1.2-1.6-fold stimulation) and no enhancement for native gizzard tropomyosin. Neither the presence of caldesmon nor native gizzard tropomyosin induced detectable binding of the amino-terminal deletion mutant rCSM beta-7 to F-actin. These results clearly imply the importance of the amino-terminal 7 amino-acid residues of CSM beta in the actin binding and the caldesmon enhancement.

Structure and complete nucleotide sequence of the gene encoding rat fibroblast tropomyosin 4.

We have isolated and determined the complete nucleotide sequence of the gene that encodes the 248 amino acid residue fibroblast tropomyosin, TM-4. The TM-4 sequence is encoded by eight exons, which span approximately 16,000 bases. The position of the intron-exon splice junctions relative to the final transcript are identical to those present in other vertebrate tropomyosin genes and the Drosophila melanogaster TMII gene. We have found no evidence that the rat TM-4 gene is alternatively spliced, unlike all the other tropomyosin genes from multicellular organisms that have been described. Typical vertebrate tropomyosin genes contain some, or all, of alternatively spliced exons 1a and 1b, 2a and 2b, 6a and 6b, and 9a, 9b, 9c and 9d in addition to common exons 3, 4, 5, 7 and 8. The rat fibroblast TM-4 mRNA is encoded by sequences most similar to exons 1b, 3, 4, 5, 6b, 7, 8 and 9d. Two exon-like sequences that are highly similar to alternatively spliced exons 2b and 9a of the rat beta-tropomyosin gene and the human TMnm gene have been located in the appropriate region of the gene encoding rat fibroblast TM-4. However, several mutations in these sequences render them non-functional as tropomyosin coding exons. We have termed these exon-like sequences, vestigial exons. The evolutionary relationship of the rat TM-4 gene relative to other vertebrate tropomyosin genes is discussed.

Differential effects of various phosphodiesterase inhibitors, pyrimidine and purine compounds, and inorganic phosphates on cyclic CMP, cyclic AMP and cyclic GMP phosphodiesterases.

The effects of various compounds on homogeneous cyclic CMP phosphodiesterase (cyclic CMP-PDE) from pig liver were compared with the effects on cyclic AMP phosphodiesterase (cyclic AMP-PDE) and cyclic GMP phosphodiesterase (cyclic GMP-PDE). Of the conventional inhibitors for AMP-PDE and cyclic GMP-PDE, only Sch 15280 was found to inhibit cyclic CMP-PDE. Nucleoside monophosphates, orthophosphate, and 2':3'-cyclic nucleotides were rather specific and were more effective in inhibiting cyclic CMP-PDE, compared to their effects on cyclic AMP-PDE and cyclic GMP-PDE. On the other hand, nucleoside di-and triphosphates and pyrophosphate (PPi) were less effective in inhibiting cyclic CMP-PDE and were without marked effect on cyclic AMP-PDE and cyclic GMP-PDE. Orthophosphate (Pi) was more potent than CMP, CDP and CTP in inhibiting cyclic CMP-PDE, with a rank order of inhibitory potency of Pi greater than CMP greater than CDP greater than CTP. Of the 3' :5'-cyclic nucleotides examined, cyclic UMP was more specific in inhibiting cyclic CMP-PDE compared to its effect on cyclic AMP-PDE and cyclic GMP-PDE. In all experiments similar results were obtained when either cyclic CMP or cyclic AMP was used as a substrate for this multifunctional cyclic CMP-PDE, supporting the contention that a single catalytic site on the enzyme is responsible for the hydrolysis of both cyclic CMP and cyclic AMP. The present studies further support our original suggestion that cyclic CMP-PDE is a unique enzyme that is distinguishable from the conventional enzymes for purine cyclic nucleotides.

Regulation of the neuron-specific exon of clathrin light chain B.

Clathrin light chain B (LCB) is a major component of clathrin coated vesicles, which are structures involved in intracellular transport. A neuron-specific isoform of LCB is generated by incorporation of a single exon (EN) using an alternative splicing mechanism that reflects the special demands of neurons, such as axonal transport and synaptic neurotransmission. Here, we demonstrate that this neuron-specific exon is developmentally regulated and is excluded in non-neuronal cells because its 5' and 3' splice sites deviate from the mammalian consensus sequences. A gel retardation assay indicated the presence of a developmentally regulated factor in brain that binds to the neuronal exon. In addition, EN usage is repressed by increasing the concentration of htra2-beta1, a splice factor whose isoform expression is influenced by neuronal activity. We propose that a brain-specific factor is involved in EN recognition during development and adulthood. In addition, ubiquitously expressed splicing factors such as htra2-beta1 are involved in regulating EN expression in the adult brain.

Thymus cell variations in AKR leukemia.

The thymus cells from spontaneous and 1st generation AKR leukemic mice were investigated, using electronic cell-volume distributions. The growth pattern of thymus cells in the mice with transplanted leukemic cells varied with the type of donor cells injected. After the transplantation of leukemic cells into 2 month-old recipients, the cortex of thymus decreased in size and medulla of thymus enlarged. The thymus underwent atrophy next, followed by a proliferation of the large-sized cells (greater than channel 40, 136 micrometer3). The electronic cell-volume distributions of the thymus cells from the spontaneous leukemic mice encompass the distributions of thymus cells during the growth of the transplanted leukemia.

The Ras-ERK pathway modulates cytoskeleton organization, cell motility and lung metastasis signature genes in MDA-MB-231 LM2.

MDA-MB-231 LM2 (herein referred to as LM2) is a derivative of MDA-MB-231 cells that was selected for its ability to metastasize to lung tissue in vivo. We investigated cellular properties of LM2 including actin cytoskeleton organization, motility and signaling pathways that drive the expression of genes associated with the lung metastasis signature. Parental cells exhibit well-developed stress fibers, whereas LM2 had poorly organized stress fibers. LM2 exhibited higher levels of K-Ras protein and corresponding higher levels of phosphorylated ERK compared with parental cells. The Ras-ERK pathway was responsible for the disruption of stress fibers because inhibition of MEK with UO126 or small interfering RNA (siRNA) against K-Ras or ERK1/2 resulted in restoration of stress fibers and focal adhesions. We observed that the K-Ras-ERK pathway is important for the expression of genes associated with the lung metastasis signature. Paradoxically, inhibition of the Ras-ERK pathway did not result in inhibition of cell motility but was accompanied by activation of the phosphatidylinositol 3-kinase (PI3K) pathway. Inhibition of both ERK and PI3K pathways was required to inhibit motility of LM2 cells. These results suggest that both ERK and PI3K pathways drive motile functions of metastatic LM2 cells and genes associated with the lung metastasis signature.