Effect of Mts1 on the structure and activity of nonmuscle myosin II.

The mts1 gene codes for a 9 kDa protein belonging to the S100 subfamily of Ca2+-binding proteins and is known to play a role in metastasis. Its role in metastasis may be through cellular locomotion, as transfection of mts1 into mouse mammary adenocarcinoma cells increases cellular motility in modified Boyden chemotaxis chambers. The Mts1 protein interacts with nonmuscle myosin II in the presence of Ca2+ with an affinity of approximately 7.9 x 10(4) M-1 and an approximate stoichiometry of 3 mol of Mts1/mol of myosin heavy chain. No interaction was found with myosin I or myosin V. The binding site of Mts1 on myosin is in the rod region, particularly to the light meromyosin portion of the rod. To understand the mechanism by which Mts1 alters cellular motility, we examined its effect on myosin structure and activity. Cosedimentation analysis and electron microscopy suggest that Mts1 destabilizes myosin filaments. In the presence of Ca2+, Mts1 inhibits the actin-activated MgATPase activity of myosin in vitro. The data demonstrate an effect of Mts1 on both myosin structure and function, and suggest a route through which Mts1 affects motility as well as metastasis.

Expression of the C terminus of the amyloid precursor protein alters growth factor responsiveness in stably transfected PC12 cells.

The amyloid precursor protein (APP) is a molecule centrally involved in Alzheimer disease pathology, but whose normal function is still poorly understood. To investigate the consequences of increased intracellular production of various regions of APP on cellular physiology, we stably transfected PC12 cells with the C-terminal 100 amino acids of the human APP. In eight transfected clones that express the APP(C100) protein, exposure to nerve growth factor (NGF) did not promote differentiation. Transfectants continued to divide and failed to elaborate extensive neurites, whereas control PC12 cells, mock-transfected PC12 cells, and a nonexpressing transfected cell line did develop neurites and stopped dividing after NGF stimulation. Unlike NGF treatment, treatment with basic fibroblast growth factor profoundly accelerated neurite outgrowth in transfected cells. Also, a dramatic increase in a tyrosine phosphatase activity was noted. Expression and accumulation of APP C100 protein in PC12 cells results in an abnormal response to growth factor stimulation.

Expression of Mts1, a metastasis-associated gene, increases motility but not invasion of a nonmetastatic mouse mammary adenocarcinoma cell line.

The mts1 gene codes for a 101 amino acid protein belonging to the S100 subfamily of Ca(2+)-binding proteins. Mts1 is overexpressed in metastatic cancers as compared to their nonmetastatic counterparts, and although mts1 is known to be involved in the metastatic phenotype (Davies et al., 1993; Grigorian et al., 1993), the role mts1 plays in this process is not clearly understood. In order to determine what role mts1 plays in the process of metastasis, we have performed transfection studies on nonmetastatic and metastatic mouse mammary adenocarcinoma cell lines, CSML0 and CSML100, respectively (Senin et al., 1983, 1984). The metastatic variant, CSML100, expresses high levels of mts1, whereas the nonmetastatic variant, CSML0, expresses almost no mts1. CSML0 cells transfected with mts1 were assessed in in vitro motility and invasion assays, as well as in vivo metastasis assays to determine the role of mts1 in these processes. Cell lines expressing mts1 display an altered morphology as well as increased motility in modified Boyden chemotaxis chambers. However, no significant increase in in vitro invasion or in in vivo metastasis was observed. Therefore, the presence of mts1 may be important for metastasis by increasing motility, but may not be sufficient for invasion in vitro or metastasis in vivo. Very low levels of type IV collagenase activities were observed in CSML0 cells and the transfectants, as opposed to the highly metastatic CSML100 cells, where high levels of type IV collagenase activities were observed. It is possible that the presence of these proteases in addition to mts1 may be responsible for the high metastatic potential of the CSML100 in vivo.

Interaction of metastasis associated Mts1 protein with nonmuscle myosin.

The mts1 gene codes for a 101 amino acid protein which belongs to the subfamily of S100 Ca(2+)-binding proteins and is overexpressed in metastatic cancers as compared to their nonmetastatic counterparts. While the Mts1 protein is putatively involved in cytoskeletal-membrane interactions, its exact physiological role is not known. In order to gain insight into the biological function of Mts1, its expression was monitored throughout mouse embryogenesis as well as in normal adult mouse tissues. In situ hybridizations of mouse embryos showed expression of mts1 mRNA to be highest in trophoblast cells of a day 8 old embryo. In normal adult mouse tissues, immunocytochemistry revealed Mts1 expression in a T-cell specific pattern in both the thymus and spleen. These results indicate that the expression of mts1 is prevalent in motile cell types, and to further elucidate the role of Mts1, gel overlay and GST-fusion protein experiments were performed to identify proteins that interact with the Mts1 protein. Nonmuscle myosin II was found to interact with Mts1, suggesting a role for Mts1 in the process of motility and lending credence to the hypothesis that Mts1 may enhance metastatic potential by increasing the motility of cancerous cells.

NMDA and AMPA receptors in transgenic mice expressing human beta-amyloid protein.

The human beta-amyloid protein may play an important, possibly primary, role in the pathogenesis of Alzheimer's disease (AD), and it appears to potentiate the susceptibility of neurons to excitotoxicity. AD is associated with alterations in the N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) subtypes of glutamate receptors, and it has been suggested that excitotoxicity may play a role in neuronal damage in AD. In this study, we have used quantitative receptor autoradiography to examine NMDA and AMPA receptors in transgenic mice that contain the gene for the carboxyl-terminal 100 amino acids of the human amyloid precursor protein, beginning with the beta-amyloid region, which is under the control of the JC viral early region promoter. Reverse transcriptase-polymerase chain reaction confirmed that the brains of transgenic mice expressed beta-amyloid mRNA and that control mice did not. NMDA receptors, assessed with [3H]MK-801, were unchanged in the transgenic compared with the control mice. In the transgenic mice, there were no significant changes in [3H]AMPA receptor binding compared with controls. This study represents the first attempt to evaluate in transgenic mice the in vivo interaction between beta-amyloid expression and excitatory amino acid receptors.

Expression of the human beta-amyloid protein of Alzheimer's disease specifically in the brains of transgenic mice.

The human beta-amyloid protein is deposited in senile plaques and in the cerebro-vasculature of people with Alzheimer's disease and Down's syndrome. The precise role of beta-amyloid in Alzheimer's disease pathology is presently unknown. To study the properties of beta-amyloid in vivo, we generated transgenic mice that harbor the gene for the carboxyl-terminal 100 amino acids of the human amyloid precursor protein, beginning with the beta-amyloid region, under control of the JC viral early region promoter. The mRNA is expressed exclusively in brain tissue. Further, we demonstrate increased levels of beta-amyloid immunoreactivity on fixed brain tissue. These animals will be useful as a model to study beta-amyloid deposition and its consequences.

Alzheimer cortical neurons containing abundant amyloid mRNA. Relationship to amyloid deposition and senile plaques.

Since the detailed molecular events leading to the formation of amyloid-containing senile plaques of the Alzheimer's disease (AD) brain are incompletely understood, the present studies were undertaken to address this issue using a combination of molecular and cytochemical approaches. Amyloid precursor protein riboprobes containing the A4 (beta-amyloid) domain were applied to cortex using the in situ hybridization method to examine the distribution of neuronal amyloid mRNA in relation to the laminar pattern of amyloid deposition and the localization of plaques. The derived data indicated that high levels of amyloid mRNA can be synthesized by AD cortical neurons that appeared to be morphologically intact. The distribution of these cells was not coincident with the cortical laminar pattern that is typical of amyloid deposits observed after immunostaining with anti-A4 monoclonal antibodies. Further, there was no obvious relationship between neurons containing abundant amyloid mRNA and the distribution of plaques identified by thioflavin S staining. While the neuronal synthesis of amyloid may be a significant factor at some point during plaque formation, it may not be the exclusive determinant. The possibility is raised that processes affecting secretion, diffusion, and/or transport of amyloid away from neuronal or non-neuronal cells of origin to sites of deposition may be meaningful aspects of the molecular pathology of Alzheimer's disease.

Requirement for proliferating cell nuclear antigen expression during stages of the Chinese hamster ovary cell cycle.

Proliferating cell nuclear antigen (PCNA/cyclin) is a nuclear protein that can stimulate purified DNA polymerase delta in vitro, and its synthesis correlates with the proliferation rate of cells. We have attempted to determine whether synthesis of PCNA/cyclin in Chinese hamster ovary cells is necessary to regulate entry into S phase. We have measured cellular PCNA/cyclin concentration of the mRNA or protein throughout the cell cycle. Cells were separated by centrifugal elutriation into populations enriched for G-1, S, and G-2/M phases. Quantitative Northern hybridization analysis was performed on RNA isolated from each cell population by using a cDNA clone of PCNA/cyclin as a probe. Results demonstrated that although intact PCNA/cyclin mRNA is present during all phases of the cell cycle, an induction of about 3-fold occurs during S phase. Two-parameter staining for PCNA/cyclin and DNA, and analysis by flow cytometry, confirmed that the quantity of PCNA/cyclin protein in the cells increases severalfold in G-1 or early S phase but generally is invariant in S and G-2/M phases. This cell cycle dependence of PCNA/cyclin expression suggests that the observed synthesis is a prerequisite for initiation of DNA replication. Introduction of an antisense oligonucleotide complementary to the PCNA/cyclin mRNA to inhibit PCNA/cyclin synthesis effectively prevented entry of G-1 phase cells into S phase. A complementary sense oligonucleotide used as a control did not have an inhibitory effect. This result suggests that a threshold concentration of PCNA/cyclin is necessary for entry into S phase.

Overexpression of amyloid precursor protein A4 (beta-amyloid) immunoreactivity in genetically transformed cells: implications for a cellular model of Alzheimer amyloidosis.

Among the major obstacles to clarifying molecular mechanisms involved in amyloid metabolism in Alzheimer disease has been the unavailability of laboratory models for this uniquely human disorder. The present studies were aimed at establishing genetically engineered cell lines that overexpress amyloid immunoreactivity and that may be relevant to amyloid accumulation in the Alzheimer disease brain. We used cloned amyloid cDNA that contains a region encoding A4 (beta-polypeptide) amino acids along with recently developed tumor virus vectors derived from simian virus 40 to prepare transformed cells. After transient and permanent transfection, a variety of cell types overexpressed A4 immunoreactivity that was detected by highly specific monoclonal antibodies. We observed that the use of an amyloid subdomain containing the A4 region, but lacking the sequence of a Kunitz-type protease inhibitor found in amyloid precursor protein variants, was sufficient to obtain cells that overproduced an A4 epitope. The transformed cells were readily propagated in culture and may provide an experimental medium to elucidate aspects of the molecular pathogenesis of Alzheimer disease. The cellular models may also serve as tools for deriving potentially useful therapeutic agents.

Preparation of a recombinant cDNA library from poly(A+) RNA of the Alzheimer brain. Identification and characterization of a cDNA copy encoding a glial-specific protein.

Studies were undertaken to assess the extent to which messenger RNA prepared from the postmortem Alzheimer's disease (AD) brain can be used for the successful preparation of a recombinant cDNA library. Initial experiments focused on the glial-specific marker glial fibrillary acidic protein (GFAP) since GFAP expression appeared to be a model for further studies on mRNAs that may continue to be expressed at high levels in the vicinity of lesioned sites in the AD brain. An AD cDNA library, prepared in the lambda gt11 expression vector system contained GFAP-specific recombinants. One of these was sequenced and the insert was shown to exhibit 88% homology with the similar sequence from mouse GFAP. As established by Northern blots, the size of the GFAP mRNA prepared from the routinely acquired postmortem AD cortex, approximately 2.7 kb, was the same as from a neurologically normal control brain. These results agree with earlier studies on GFAP mRNA from fresh mouse brain. The results demonstrate that in the postmortem AD brain, astroglial-specific mRNA remains sufficiently stable for molecular genetic analysis and may serve as a useful model for examining the genetic expression of mRNAs that may be related to the molecular pathogenesis and the etiology of AD.

The postmortem Alzheimer brain is a source of structurally and functionally intact astrocytic messenger RNA.

Although the precise role of astrocytes in the pathogenesis of Alzheimer's disease (AD) is currently undefined, studies carried out at the molecular level may lead to new insights into the functioning of this class of brain cells in dementia. In order to facilitate such investigations, methods are described that establish that structurally and functionally intact messenger RNA (mRNA) for an astrocytic marker, glial fibrillary acidic protein (GFAP), is present in the postmortem Alzheimer's disease brain after long postmortem intervals. Rapid preparative procedures were used to obtain poly(A)+ RNA from postmortem control and AD cortices. In vitro protein synthesis was carried out in a reticulocyte system. Relative to controls, AD mRNA synthesized a two-fold higher level of a 50,000 mol.wt. protein that was immunologically identified as GFAP. High levels of GFAP synthesis by purified mRNA from AD cortices was independent of age at death and postmortem interval up to 24 h. Northern blot hybridization using a cloned human GFAP riboprobe was used to evaluate postmortem GFAP mRNA stability. No appreciable degradation products of GFAP mRNA were observed on Northern blots for at least 10 h postmortem in poly(A)+ RNA extracted from the AD brain. The described methodology demonstrates that the postmortem AD brain is an excellent source of functionally and structurally intact astrocyte-specific mRNA.

Molecular cloning of amyloid cDNA derived from mRNA of the Alzheimer disease brain: coding and noncoding regions of the fetal precursor mRNA are expressed in the cortex.

To gain insight into factors associated with the excessive accumulation of beta-amyloid in the Alzheimer disease (AD) brain, the present studies were initiated to distinguish between a unique primary structure of the AD-specific amyloid precursor mRNA vis a vis other determinants that may affect amyloid levels. Previous molecular cloning experiments focused on amyloid derived from sources other than AD cases. In the present work, we cloned and characterized amyloid cDNA derived directly from AD brain mRNA. Poly(A)+ RNA from AD cortices was used for the preparation of lambda gt11 recombinant cDNA libraries. An insert of 1564 nucleotides was isolated that included the beta-amyloid domain and corresponded to 75% of the coding region and approximately equal to 70% of the 3'-noncoding region of the fetal precursor amyloid cDNA reported by others. On RNA blots, the AD amyloid mRNA consisted of a doublet of 3.2 and 3.4 kilobases. In control and AD cases, the amyloid mRNA levels were nonuniform and were independent of glial-specific mRNA levels. Based on the sequence analysis data, we conclude that a segment of the amyloid gene is expressed in the AD cortex as a high molecular weight precursor mRNA with major coding and 3'-noncoding regions that are identical to the fetal brain gene product.

More precise localization of the human factor IX gene by in situ hybridization.

The location of the human antihemophilic Factor IX has been more specifically assigned from the region Xq27----qter to Xq26----q27 by quantitative in situ hybridization. The present study utilized a complex hybridization probe and prephotographed G-banded human chromosomes to improve analytical sensitivity and accuracy.