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Acute leukemias arise secondary to chromosomal aberrations that cause dysfunctions in gene regulation and regulatory factors. Significant differences in morphology between acute leukemic and nonleukemic hematopoietic cells are readily observed. How morphologic changes of the nuclei of acute leukemic cells relate to the underlying functional alterations of gene expression is minimally understood. Spatial modifications in the representation and/or organization of regulatory factors may be functionally linked to perturbations of gene expression in acute leukemic cells. Using in situ immunofluorescence microscopy, we addressed the interrelationships of modifications in nuclear morphology with the intranuclear distribution of leukemia-related regulatory factors (including ALL-1, PML, and AF-9) in cells from patients with acute leukemia. We compared the localization of leukemia-associated proteins with various factors involved in gene transcription and RNA processing (e.g., RNA polymerase II and SC-35). Our findings suggest that there are leukemia-associated aberrations in mechanisms that direct regulatory factors to sites within the nucleus. This misplacement of key cognate factors may contribute to perturbations in gene expression characteristic of leukemias.

Evidence is accruing that the architectural organization of nucleic acids and regulatory proteins within the cell nucleus support functional interrelationships between nuclear structure and gene expression. The punctate distribution of several transcription factors has provided several paradigms for pursuing mechanisms that direct these regulatory proteins to subnuclear sites where gene activation or suppression occurs. Sequences that are necessary and sufficient to direct regulatory proteins to transcriptionally active nuclear domains have been identified. Mutations that disrupt intranuclear targeting signals lead to modified subnuclear distribution of transcription factors and aberrant expression in tumor cells. J. Cell. Biochem. Suppls. 32/33:24-31, 1999.

Functional interrelationships between components of nuclear architecture and control of gene expression are becoming increasingly evident. There is growing appreciation that multiple levels of nuclear organization integrate the regulatory cues that support activation and suppression of genes as well as the processing of gene transcripts. The linear organization of genes and promoter elements provide the potential for responsiveness to physiological regulatory signals. Parameters of chromatin structure and nucleosome organization support synergism between activities at independent regulatory sequences and render promoter elements accessible or refractory to transcription factors. Association of genes, transcription factors, and the machinery for transcript processing with the nuclear matrix facilitates fidelity of gene expression within the three-dimensional context of nuclear architecture. Mechanisms must be defined that couple nuclear morphology with enzymatic parameters of gene expression. The recent characterization of factors that mediate chromatin remodeling and intranuclear targeting signals that direct transcription factors to subnuclear domains where gene expression occurs, reflect linkage of genetic and structural components of transcriptional control. Nuclear reorganization and aberrant intranuclear trafficking of transcription factors for developmental and tissue-specific control that occurs in tumor cells and in neurological disorders provides a basis for high resolution diagnostics and targeted therapy. J. Cell. Biochem. Suppls. 30/31:220-231, 1998. © 1998 Wiley-Liss, Inc.

The relationship of cell proliferation to the temporal expression of genes characterizing a developmental sequence associated with bone cell differentiation was examined in primary diploid cultures of fetal calvarial derived osteoblasts by the combined use of autoradiography, histochemistry, biochemistry, and mRNA assays of osteoblast cell growth and phenotypic genes. Modifications in gene expression define a developmental sequence that has 1) three principle periods--proliferation, extracellular matrix maturation, and mineralization--and 2) two restriction points to which the cells can progress but cannot pass without further signals--the first when proliferation is down-regulated and gene expression associated with extracellular matrix maturation is induced, and the second when mineralization occurs. Initially, actively proliferating cells, expressing cell cycle- and cell growth-regulated genes, produce a fibronectin/type I collagen extracellular matrix. A reciprocal and functionally coupled relationship between the decline in proliferative activity and the subsequent induction of genes associated with matrix maturation and mineralization is supported by 1) a temporal sequence of events in which there is an enhanced expression of alkaline phosphatase immediately following the proliferative period, and later, an increased expression of osteocalcin and osteopontin at the onset of mineralization; 2) increased expression of a specific subset of osteoblast phenotype markers, alkaline phosphatase and osteopontin, when proliferation is inhibited by hydroxyurea; and 3) enhanced levels of expression of the osteoblast markers as a function of ascorbic acid-induced collagen deposition, suggesting that the extracellular matrix contributes to both the shutdown of proliferation and the development of the osteoblast phenotype.

When human lymphocytes are treated with concanavalin-A (con A) and hemocyanin, the hemocyanin marker, which demonstrates con A binding sites, can be visualized by scanning (SEM) and transmission electron microscopy (TEM) on both critical-point-dried and freeze-dried cells. The ability to visualize the hemocyanin marker by SEM, its quantity and distribution, were all similar in lymphocytes prepared by both drying procedures. By TEM, hemocyanin was seen adjacent to the plasma membrane on critical-point-dried lymphocytes. In contrast, freeze-dried cells showed hemocyanin labeling at some distance from the plasma membrane (40-70 nm) as well as adjacent to it. The distribution of hemocyanin corresponded to the thickness of the amorphous coat seen on fixed, freeze-dried cells. Therefore, the extracellular coat on freeze-dried lymphocytes is a carbohydrate-containing glycocalyx.

A quantitative comparison of isolated lymphocytes prepared for SEM by the critical-point drying (CPD) and freeze drying (FD) methods revealed that the mean cellular diameter dropped from 7.9 micron after fixation to a final diameter 6.9 micron after FD, and to 5.7 micron after CPD. In addition to their larger sizes, FD lymphocytes were immediately distinguishable by their more complex surfaces, featuring wider microvilli which often emanated from ridges on the cell surface and branched extensively near their bases. The mean width of microvilli was 0.22 micron after FD and 0.12 micron after CPD. The number of microvilli per cell was essentially the same by the two methods. In view of these findings, a critical comparison of the CPD and FD methods using the particular cells or tissue to be investigated is an essential prelude to a rigorous SEM study.