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S K Hanks

Publications and source records attributed to S K Hanks.

45 records · Page 3Linked to original sources
PubMed1986

Proto-oncogene fos: an inducible multifaceted gene.

Proto-oncogene fos, which is expressed during cell growth and cell differentiation and development, is a multifaceted gene. The viral homolog, v-fos, was identified as the resident transforming gene of FBJ-murine osteosarcoma virus, which induces bone tumors in mice. Owing to an in-frame deletion during the biogenesis of the v-fos gene, the products of viral and cellular fos proteins differ at their C-termini. Despite different C-termini, both fos proteins are nuclear in their location and can transform fibroblasts in vitro. However, transformation by the c-fos gene requires removal of a 67-base pair sequence from the 3' noncoding domain. Proto-oncogene fos is a highly inducible gene in response to a variety of growth factors and differentiation-specific inducers. The expression of the fos gene is not modulated during the cell cycle.

Animals↗
PubMed1985

Early initiation of DNA synthesis in G1 phase HeLa cells following fusion with red cell ghosts loaded with S-phase cell extracts.

A modification of polyethylene glycol-mediated cell fusion procedure has been described and standardized for red blood cell mediated microinjection of proteins into cells in suspension. Using this procedure, proteins are routinely introduced into 50% of the target cells. We have applied this microinjection procedure to introduce cytoplasmic extracts obtained from S-phase HeLa cells into HeLa cells synchronized in G1. This experiment resulted in an accelerated entry of the G1 cells into S phase as measured by the incorporation of [3H]thymidine. This technique may provide a means to identify and characterize the S-phase factors responsible for the induction of DNA synthesis.

Cell Cycle↗
PubMed1984

The ultrastructural organization of prematurely condensed chromosomes.

In an effort to understand the arrangement of the basic 30 nm chromatin fibre within metaphase chromosomes, changes in the organization of prematurely condensed chromosomes (PCC) were examined as a function of progression through the cell cycle. The structural features of PCC observed under the light microscope were compared with those obtained by scanning electron microscopy. PCC with varying levels of condensation were obtained by fusing mitotic HeLa cells with interphase cells synchronized at different times in the cell cycle. PCC from G1 cells are composed of rather tightly packed bundles of tortuous chromatin fibres. The density of fibre packing along the longitudinal axis of G1-phase PCC is lower and less uniform than that of metaphase chromosomes. Early G1 PCC exhibit gyres suggesting a despiralized chromonema. The condensed domains in G1 PCC appear to be organized as supercoiled loops; whereas fibre-sparse domains consist of longitudinal fibres running along the chromosome axis. As cells progressed towards S phase, a greater proportion of highly extended regions containing prominent longitudinal fibres became evident in the PCC. The pulverized appearance of S-phase PCC under the light microscope corresponded to the highly condensed, looping fibre domains separated by more extended segments containing longitudinal fibres that are visualized using the scanning electron microscope. Active sites of DNA synthesis are implicated to be localized within extended longitudinal fibres. Post-replicative chromosome maturation extends through the G2 period and appears to involve rearrangement of the extended longitudinal fibres into packed looping-fibre clusters, which then coalesce. These observations support the model for packing DNA into chromosomes proposed in 1980 by Mullinger & Johnson. Briefly, this model suggests that the chromonema of each metaphase chromatid contains regions composed of folded longitudinal chromatin fibres as well as looping fibres that emerge from the axis at distinct foci. The final level of chromatin packing in metaphase chromosomes is attained by spiralization of the chromonema.

Chromatin↗
PubMed1983

Relationship between histone phosphorylation and premature chromosome condensation.

The relationship between histone phosphorylation and chromosome condensation was investigated by determining changes in phosphorylation levels of histones H1 and H3 following fusion between mitotic and interphase cells and subsequent premature chromosome condensation. We detected significant increases in the levels of phosphorylation of H1 and H3 from interphase chromatin in which a majority of nuclei had undergone premature chromosome condensation. In addition, we noted significant decreases in the phosphate content of the highly phosphorylated mitotic H1 and H3, presumably resulting from phosphatase activity contributed by the interphase component of mitotic/interphase fused cells. These observations further strengthen the correlation between histone phosphorylation and the changes in chromosome condensation associated with the initiation of mitosis. This study also suggests that maintenance of the mitotic chromosomes in a highly condensed state does not require the continued presence of histones in a highly phosphorylated form.

Cell Nucleus↗
PubMed1983

Cell cycle-specific changes in the ultrastructural organization of prematurely condensed chromosomes.

Prematurely condensed chromosomes (PCC) of HeLa cells synchronized in different phases of the cell cycle were analyzed by high-resolution scanning electron microscopy. The purpose of this study was to examine changes in the arrangement of the basic 30-nm chromatin fiber within interphase chromosomes associated with progression through the cell cycle. These studies revealed that highly condensed metaphase chromosomes and early G1-PCC consisted of tightly packed looping fibers. Early to mid G1-PCC were more extended and exhibited gyres suggestive of a despiralized chromonema. Further attenuation of PCC during progression through G1 was associated with a gradual transition from packed looping fibers to single extended longitudinal fibers. This process occurs prior to the initiation of DNA synthesis which appears to be localized within single longitudinal fibers. Following replication of a chromosome segment, extended longitudinal fibers were rapidly reorganized into packed looping fiber clusters concomitant with the formation of a multifibered chromosome axis. This results in the characteristic "pulverized" appearance of S-PCC when viewed by light microscopy. Subsequently, adjacent looping fiber domains coalesce, resulting in the uniformly packed, looping fiber arrangement observed in G2-PCC. Spiralization of the chromonema during the G2-mitotic transition results in the formation of highly compact metaphase chromosomes.

Cell Cycle↗
PubMed1980

Chromatin structure during the prereplicative phases in the life cycle of mammalian cells.

The object of this study was to determine the kinetics of chromosome decondensation during the G1 period of the HeLa cell cycle. HeLa cells synchronized in the G1 period following the reversal of mitotic block were fused with Colcemid-arrested mitotic HeLa cells at 1.5, 3, 5, and 7 h after the reversal of N2O block. The resulting prematurely condensed chromosomes (PCC) were classified into six categories depending on the degree of their condensation. The frequency of occurrence of each category was plotted as a function of time after mitosis. The results of this study indicate that the process of chromosome decondensation, initiated during the telophase of mitosis continues throughout the G1 period without any interruption, thus the chromatin reaches an ultimate state of decondensation by the end of G1 period, when DNA synthesis is initiated.

Chromatin↗
PubMed1980

Initiation of DNA synthesis in the prematurely condensed chromosomes of G1 cells.

The objective of this study was to investigate whether G1 cells could enter S phase after premature chromosome condensation resulting from fusion with mitotic cells. HeLa cell synchronized in early G1, mid-G1, late G1, and G2 and human diploid fibroblasts synchronized in G0 and G1 phases were separately fused by use of UV-inactivated Sendai virus with mitotic HeLa cells. After cell fusion and premature chromosome condensation, the fused cells were incubated in culture medium containing Colcemid (0.05 micrograms/ml) and [3H]thymidine ([3H]ThdR) (0.5 microCi/ml; sp act, 6.7 Ci/mM). At 0, 2, 4, and 6 h after fusion, cell samples were taken to determine the initation of DNA synthesis in the prematurely condensed chromosomes (PCC) on the basis of their morphology and labeling index. The results of this study indicate that PCC from G0, G1, and G2 cells reach the maximum degree of compaction or condensation at 2 h after PCC induction. In addition, the G1-PCC from normal and transformed cells initiated DNA synthesis, as indicated by their "pulverized" appearance and incorporation of [3H]ThdR. Further, the initiation of DNA synthesis in G1-PCC occurred significantly earlier than in the mononucleate G1 cells. Neither pulverization nor incorporation of label was observed in the PCC of G0 and G2 cells. These findings suggest that chromosome decondensation, although not controlling the timing of a cell's entry into S phase, is an important step for the initiation of DNA synthesis. These data also suggest that the entry of a S phase may be regulated by cell cycle phase-specific changes in the permeability of the nuclear envelope to the inducers of DNA synthesis present in the cytoplasm.

Cell Cycle↗
PubMedDate not supplied

Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase.

The cytoplasmic focal adhesion protein-tyrosine kinase (FAK) localizes with surface integrin receptors at sites where cells attach to the extracellular matrix. Increased FAK tyrosine phosphorylation occurs upon integrin engagement with fibronectin. Here we show that adhesion of murine NIH3T3 fibroblasts to fibronectin promotes SH2-domain-mediated association of the GRB2 adaptor protein and the c-Src protein-tyrosine kinase (PTK) with FAK in vivo, and also results in activation of mitogen-activated protein kinase (MAPK). In v-Src-transformed NIH3T3, the association of v-Src, GRB2 and Sos with FAK is independent of cell adhesion to fibronectin. The GRB2 SH2 domain binds directly to tyrosine-phosphorylated FAK. Mutation of tyrosine residue 925 of FAK (YENV motif) to phenylalanine blocks GRB2 SH2-domain binding to FAK in vitro. Our results show that fibronectin binding to integrins on NIH3T3 fibroblasts promotes c-Src and FAK association and formation of an integrin-activated signalling complex. Phosphorylation of FAK at Tyr 925 upon fibronectin stimulation creates an SH2-binding site for GRB2 which may link integrin engagement to the activation of the Ras/MAPK signal transduction pathway.

3T3 Cells↗