Sheldon Penman, Ph.D. - US grants

Epithelial cells, growing in tissue-like colonies, have marked and characteristic morphological and biochemical responses to both tumor promoters and to transformation that are similar in many ways. The highly differentiated MDCK line is extremely sensitive to tumor promoting agents. A characteristic morphological signature results from exposure to promoters that is visualized with fluorescent anti-cytokeratin antibodies and by video-enhanced phase microscopy. Within 15 minutes, the stationary epithelial colonies are converted to clusters of independent and highly motile cells. Quantitative morphometric analysis of the cell response to various promoters will measure the rate of change of: a) total inter-nuclear spacing, b) nuclear shape and c) integrated curvature of cellular processes within the colonies as a function of promoter dose. Analysis will use a simple graphics tablet and a small computer. We will correlate the in vitro response with promoter efficacy, as observed in vivo, to determine if our measurements constitute a short-term test for complete and second stage promoters. Permanent transformation by a single dose of a complete carcinogen or by infection with Moloney sarcoma virus results in a morphological signature and an altered metabolic response similar to that induced by tumor promoters. The metabolic response to an altered cell shape is also markedly changed. A transfection system will be established with MDCK in order to insert several activated oncogenes in addition to v-mos, some of which should give similar morphological and biochemical responses. Complete carcinogens give a prompt response which in some ways resembles that of promoters and the possibility of the promotion-like activity resulting from protein alkylation by these agents will be tested using ultimate carcinogens. Phosphorylation changes, including those specific to tyrosine, in the presence of TPA have been noted by others and we find these to be located principally in the nuclear matrix. We will examine phosphorylation in the presence of other promoters and in cells transformed by specific oncogenes. The activity of protein kinase C will be determined and the possible association of this activity with skeletal elements in promoted and transformed cells will be measured.

The nonchromatin structure of the nucleus has been prepared using a new strategy that preserves many specific associations of DNA and RNA with the matrix. Both matrix morphology and biochemical properties depend critically on the preservation of nuclear RNA and the preparation is termed the RNP-matrix. This structure selectively binds active chromatin and non-message hnRNA. We suggest that RNP-matrix plays a fundamental role in regulating patterns of gene expression and propose to study the molecular biology of its associated nucleic acids. The RNP-matrix morphology appears, in resinless sections, far better preserved than in preparation made by harsher procedures. All actively transcribed DNA sequences probed so far are tightly bound to the matrix, one third of the DNA sequences directly to the matrix proteins and the remainder linked to matrix hnRNP. HnRNA is separated into distinct classes: message precursor RNA is eluted as an RNP particle (prt-hnRNA) while non-message transcripts remain bound to the matrix (mtx-hn RNA). Cloned cDNA probes hybridize only to the prt-hnRNA while the cloned non-message HindIII l.9-kb repeat hybridizes only to mtx-hnRNA. The proteins of the RNP-matrix change radically with differentiation and serve as a marker for cell type. Transformation markedly alters matrix proteins with very different patterns resulting from onc gene transfection and from chemical carcinogenesis. (F)

DESCRIPTION: (Adapted from applicant's abstract). Embedment-free electron microscopy shows the nuclear matrix comprised of a network of 11 nm filaments with numerous embedded dense organelles. They will use two new nuclear matrix preparations, high salt and the stabilized pre- fixed matrix. They will determine the true nature of the interchromatin granules using reinless section EM of the nuclear matrix preparations immunostained with antibodies to splicing components (in collaboration with Dr. Phil Sharp laboratory). They will combine these results with in situ hybridization with intron specific probes (in collaboration with Dr.Jeanne Lawrence) to locate which nuclear matrix organelle(s) are the location of splicing. In situ hybridization of hnRNA tracks will image the RNA transport machinery. Hybridization with an intermediate repetitive sequence will identify the location of hnRNA that does not serve as mRNA precursor. They will seek monoclonal antibodies to NM filament proteins to determine their number, molecular sequences and fate at mitosis.

The nuclear matrix (NM) is emerging as a crucial element in nuclear metabolism. It organizes chromatin, serves in RNA splicing and may participate in selecting DNA regions for transcription. Despite growing interest, methods of NM preparation have been largely arbitrary and harsh. We have systematically applied biochemical analysis and resinless section electron microscopy to arrive at an NM preparation closest to the in vivo structure. The NM proteins are completely distinct from those of chromatin with almost no cross contamination of fractions. In resinless sections the matrix structure and lamina appear intact. This more nearly native NM has many proteins lost in harsher preparation, many of which are highly cell type specific. Further fractionation reveals an underlying network of core filaments which contain all of the hnRNA and serve as a structural component of the NM. The research proposed here is to continue characterizing the proteins of the NM and core filaments using MAB libraries developed in the current grant period. In this way, some NM functions can be identified. The MABs are used for spatial localization by a method developed for gold beads in resinless sections for both interphase and mitotic cells. The MABs are also used to select cDNA clones from lambda gt11 expression libraries for molecular sequencing. We have report the preliminary characterization of antigens to 9 MABs as well as results using MABs, such as to HPV 16 E7 protein, obtained from other laboratories. We have completely sequenced two NM proteins and one from the core filaments. We have also identified several different behaviors of NM proteins at mitosis and are investigating these by stereoscopic immunogold staining.

The nuclear matrix-intermediate filament (NM-IF) scaffold has been prepared using a gentle, relatively non-disruptive procedure. The cell type specific proteins of the nucleus appear largely components of the NM-IF and change markedly with tissue of origin and after with transformation by carcinogens and oncogene transfection. We are developing a library of monoclonal antibodies raised to the NM-IF from a variety of carcinoma lines and onc gene transformed cells which will be used in resinless section immunoelectron microscopy. We are examining the DNA bound specifically to the NM-IF for actively transcribed and special sequences. The prompt heat shock proteins, rpoduced by translational activation of pre-existing mRNA, are entirely bound to the NM-IF. A large subset is induced by noxious agents (e.g. arsenite) and become major NM-IF constituents and monoclonal antibodies are being raised for immunoelectron microscopy and biochemical purification. Nuclear RNA synthesis and processing will be examined in stressed cells in the presence and absence of the prompt proteins to determine a possible protective role. A subfraction of hnRNA, with the properties of mRNA precursor, is released from the NM-IF by gentle salt treatment. The remaining tightly bound hnRNP appears to play an important structural role in the NM-IF. The message-like released (m-) hnRNP will be compared to the tightly bound, presumably structural (st-) hnRNP for protein composition and content of message and repetitive sequences.

nuclear matrix; bone development; genetic regulation; cell differentiation; gene expression; osteoblasts; DNA binding protein; developmental genetics; genetic enhancer element; genetic transcription; information systems; RNase protection assay; northern blottings; laboratory rat; nuclear runoff assay; embryo /fetus cell /tissue; molecular cloning; immunocytochemistry; western blottings; gel electrophoresis; immunoelectron microscopy; electron microscopy; nucleic acid sequence; in situ hybridization;

DESCRIPTION: (Adapted from applicant's abstract). Embedment-free electron microscopy shows the nuclear matrix comprised of a network of 11 nm filaments with numerous embedded dense organelles. They will use two new nuclear matrix preparations, high salt and the stabilized pre- fixed matrix. They will determine the true nature of the interchromatin granules using reinless section EM of the nuclear matrix preparations immunostained with antibodies to splicing components (in collaboration with Dr. Phil Sharp laboratory). They will combine these results with in situ hybridization with intron specific probes (in collaboration with Dr.Jeanne Lawrence) to locate which nuclear matrix organelle(s) are the location of splicing. In situ hybridization of hnRNA tracks will image the RNA transport machinery. Hybridization with an intermediate repetitive sequence will identify the location of hnRNA that does not serve as mRNA precursor. They will seek monoclonal antibodies to NM filament proteins to determine their number, molecular sequences and fate at mitosis.