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D L Guernsey

Publications and source records attributed to D L Guernsey.

At least 19 recordsLinked to original sources
PubMed1996

Altered sensory processing in the somatosensory cortex of the mouse mutant barrelless.

Mice homozygous for the barrelless (brl) mutation, mapped here to chromosome 11, lack barrel-shaped arrays of cell clusters termed "barrels" in the primary somatosensory cortex. Deoxyglucose uptake demonstrated that the topology of the cortical whisker representation is nevertheless preserved. Anterograde tracers revealed a lack of spatial segregation of thalamic afferents into individual barrel territories, and single-cell recordings demonstrated a lack of temporal discrimination of center from surround information. Thus, structural segregation of thalamic inputs is not essential to generate topological order in the somatosensory cortex, but it is required for discrete spatiotemporal relay of sensory information to the cortex.

Animals↗
PubMed1995

Passage of X-ray-induced immortal, non-transformed phenotype by DNA-mediated transfection.

To better understand the molecular basis of X-ray-induced carcinogenesis, the immortalization step of this multistep process was examined. Primary rat embryo cells were X-irradiated in vitro and six clones were isolated. Three of these, one designated X-REF-23, were immortal and non-transformed. Transfection of high molecular weight DNA from rat X-REF-23 cells into primary mouse cells yielded two immortal and non-transformed mouse clones, 1K and 2I. Using a 2.3 kb rat-specific repetitive sequence as probe, 1K and 2I were demonstrated to contain rat DNA. This transfected DNA was not any of the known immortalization-associated proto-oncogenes. DNA from 1K was then transfected into primary mouse cells, with or without co-transfection of pSV2 neo DNA. Six immortal mouse clones were isolated and confirmed to contain rat sequences. In conclusion, the immortal phenotype can be transferred by DNA transfection.

Animals↗
PubMed1994

Detection of familial defective apolipoprotein B-100 among patients clinically diagnosed with heterozygous familial hypercholesterolemia in maritime Canada.

Familial defective apolipoprotein B-100 (FDB) is a genetic disorder resulting from a mutation in the apolipoprotein B-100 (apo B-100) gene, most frequently at position 3500, in which arginine is substituted for glutamine in the mature protein. This mutation drastically decreases the affinity of the mutant apo B-100 particle for the low-density lipoprotein (LDL) receptor, and hence decreases the clearance of cholesterol from the circulation. Familial hypercholesterolemia (FH), also a disorder of lipid metabolism, results from mutations in the gene for the LDL receptor. Both FDB and heterozygous FH occur at approximately the same frequency (1 in 500) among Caucasians and both produce clinical symptoms and signs that can be indistinguishable. Polymerase chain reaction (PCR) amplification and subsequent restriction analysis have been used to detect the substitution at codon 3500 in the apo B-100 gene using mutagenic PCR primers. At least one proband from 10 unrelated families with a history of hypercholesterolemia was screened by mutagenic PCR for FDB. Only one of 10 patients demonstrated the mutation for FDB. The mutant apo B-100 allele was shown to segregate with other clinically affected family members. These results demonstrate that molecular analysis is essential to distinguish between FDB and heterozygous FH in hypercholesterolemic families.

Adult↗
PubMed1993

Elevation of the thyroid hormone receptor erb A-alpha 2 mRNA in transformed rodent cells is due to increased message stability.

Previously, the authors reported an elevation of erb A-alpha 2 mRNAs in transformed rodent cells when compared with their non-transformed counterparts (Cancer Res., 52(1992) 2186-2190). To investigate this phenomenon further the rates of gene transcription and the effects of translation/transcription inhibition on erb A-alpha 2 mRNA expression were examined. The present study found no difference between non-transformed and transformed cells in erb A-alpha 2 gene transcription rate, nor an effect of cycloheximide on erb A-alpha 2 mRNA expression. However, a significant difference was obtained with actinomycin D. With this inhibitor, the half-life of erb A-alpha 2 mRNAs in nontransformed rodent cells was determined to be approximately 8 h. In contrast, the alpha 2 transcripts in their transformed counterparts showed no decay during this period, suggesting that the elevation of erb A-alpha 2 mRNAs in transformed rodent cells was due to increased transcript stability.

Animals↗
PubMed1992

Increased messenger RNA levels of the antagonist thyroid hormone receptor erbA-alpha 2 and decreased levels of erbA-alpha 1 and erbA-beta 1 receptor messenger RNAs in neoplastic rodent cells.

Nothern blot analysis of total RNA from the mouse C3H/10T1/2 cell line indicated that the erbA alpha gene transcribed three mRNA species of similar sizes (2.6, 5.5, 6.6 kilobases) as found in rodents. The 2.6-kilobase mRNA (erbA-alpha 2) was approximately 7- to 8-fold more abundant than either the 5.5- (erbA-alpha 1) or 6.6-kilobase species. The expression of the erbA-alpha 2 transcript increased 3- to 30-fold when "normal" mouse or rat cells were growth arrested by concluence. Triiodothyronine, at a concentration of 1 nM, had no effect on the levels of the erbA-alpha mRNA species in confluent cells nor on the levels of erbA-alpha 2 in proliferative normal or transformed C3H/10T1/2 cells. In log-phase growing cells there was a 2.5- to 5-fold increase in the relative expression of erbA-alpha 2 mRNA in transformed mouse C3H/10T1/2 cells, transformed cloned rat embryo fibroblasts (CREF), transformed rat embryo fibroblasts (REF), and a transformed temperature-sensitive rat mutant cell line (ts7E) when compared with their non-transformed counterparts. In contrast to the elevation of erbA-alpha 2 in transformed cells, erbA-alpha 1 and erbA-beta 1 mRNAs decreased in transformed mouse and rat cell lines. In conclusion, it is suggested that the increased levels of the erbA-alpha 2 transcript and the decreased levels of erbA-alpha 1 and erbA-beta 1 in neoplastic cells may account for the loss of thyroid hormone regulation of inducible pathways and decreased nuclear triiodothyronine binding as previously reported.

Animals↗
PubMed1992

Oncogenes in X-ray-transformed C3H 10T1/2 mouse cells and in X-ray-induced mouse fibrosarcoma (RIF-1) cells.

In order to better understand the molecular basis of X-ray induced carcinogenesis we have investigated RNA levels of oncogenes in an X-ray transformed C3H 10T1/2 fibroblast line (XTD) and RIF-1 cells isolated from an X-ray-induced fibrosarcoma in a C3H mouse. Steady-state levels of K-ras, H-ras, N-ras, abl, sis, src, and fos were unchanged in the X-ray-transformed cells compared with non-transformed C3H 10T1/2 cells. However, myc and raf mRNA levels were increased dramatically in the transformed cells. Data further suggests a possible alteration in processing of raf RNA in the XTD cells. Southern blot analysis of secondary transfectants induced with XTD DNA indicated that the oncogenic phenotype did not segregate with the myc or raf loci; nor with nine other oncogenes analysed.

Animals↗
PubMed1991

Altered oncogenes in UV-transformed C3H 10T1/2 mouse cells: identification of mutated H-ras allele(s).

Ultraviolet (UV) light will transform mammalian cells in culture to a phenotype which is characteristic of in vivo neoplasia. The UV-transformed C3H 10T1/2 mouse cell lines, TU-2 and TU-3, were analysed to determine the molecular mechanisms which may account for their phenotype, and to determine the types of mutations induced by UV light. DNA-transfection assays indicated that the transformed phenotype of TU-2 could not be transferred to non-transformed recipient cells. Therefore, studies were initiated to determine the mutagenic effects of UV light with respect to cellular oncogenes. Northern blot analysis indicated that five of the oncogenes analysed (erb-A, erb-B, mos, myb, and N-ras) were not expressed at detectable levels. The steady-state mRNA levels of fos, K-ras, abl, sis, and src oncogenes were similar in the C3H 10T1/2 and TU-2 cells. The mRNA levels of three oncogenes, raf, myc and H-ras, were 1.5-2.0-fold greater in the TU-2 cells compared to C3H 10T1/2. Southern blot analysis of HpaII restriction digested TU-2 DNA indicated that the H-ras oncogene has undergone methylation changes. More extensive analyses of the H-ras locus in TU-2 demonstrated a deletion of the 3' end of the gene, that may involve two separate mutated alleles. This type of damage is consistent with the lesions associated with sister chromatid exchange. While the H-ras locus in the other UV-transformed line, TU-3, showed methylation changes, there were no large genetic mutations detected by Southern blot analysis. These results suggest that UV-irradiation in vitro induces endogenous DNA damage that includes methylation changes and large genomic alterations. Further analysis will be necessary to determine the extent to which each may be involved in cell transformation.

Alleles↗
PubMed1989

Superoxide dismutase activities of differentiating clones from an immortal cell line.

We have studied superoxide dismutase (SOD) levels in the X-REF-23 rat embryo fibroblast cell line. X-REF-23 is an immortal cell line that maintains a nontransformed phenotype throughout its known lifespan. Low-passage X-REF-23 cells undergo spontaneous differentiation into muscle and adipose cells, while high-passage X-REF-23 cells undergo little or no differentiation. SOD activities were measured in subclones of X-REF-23, which differentiate into muscle (AMC subclone) or adipose (AMB-J) cells, as well as the parental nondifferentiating X-REF-23 cells. Total SOD activity increased in all three cell lines with time in culture. Cu-ZnSOD was induced in the AMB-J and the X-REF-23 cells with time in culture, whereas the AMC cells showed no induction. MnSOD activity was induced during the time period in which differentiation occurred in the two differentiating clones. In contrast, MnSOD was not induced in this time period in the nondifferentiating X-REF-23 cell line. However, MnSOD activity was induced in the latter cell line at a much later time. Levels of immunoreactive MnSOD correlated quite well with MnSOD activity in all three cell lines. The nondifferentiating X-REF-23 cells, but not the two differentiating cells lines, showed a large increase in cell organelles with time in culture. In particular, an increase in very small mitochondria was observed; these mitochondria often showed evidence of disorganization.

Adipose Tissue↗
PubMed1988

Retinoic-acid-induced modulation of c-myc not dependent on its continued presence: possible role in pre-commitment for HL-60 cells.

Induced differentiation of HL-60 human promyelocytic cells along the myeloid or monocytic lineages has been previously shown to involve an intermediate regulatory state, the pre-commitment state. Pre-commitment cells have completed the early events in the processes leading ultimately to terminal differentiation and require only an abbreviated subsequent exposure to inducer for onset of terminal differentiation. The pre-commitment state has 2 properties relevant to the present communication: (1) when induced by retinoic acid (RA), it has a characteristic duration following removal of the RA; and (2) it can also be induced by a pulse exposure to hydroxyurea. In the present studies, it was observed that after exposure of HL-60 human promyelocytic leukemia cells to RA for 24 hr (ca. one division cycle) their levels of c-myc RNA were elevated. The c-myc RNA level then remained elevated for several subsequent division cycles despite the removal of retinoic acid. Thus, retinoic acid induced a change in HL-60 c-myc RNA levels which was sustained regardless of the continued presence or absence of RA. The elevation, decreasing to control levels 3 division cycles after termination of the pulse exposure, paralleled the known duration of the pre-commitment memory state. Furthermore, a pulse exposure of HL-60 cells to a subcytotoxic dose of hydroxyurea, which is also known to induce a pre-commitment state, also induced an elevation of c-myc RNA levels. The observed changes in c-myc levels were not common to all oncogenes. C-fos responded differently to the retinoic acid treatment. Furthermore, although hydroxyurea affected c-myc levels, it did not alter c-fos levels. Most significantly, the present results suggest a cellular function for the c-myc gene product, which is derivation of the pre-commitment state.

Cell Differentiation↗
PubMed1988

Corticosterone effects on differentiation and X-ray-induced transformation of C3H/10T1/2 mouse cells.

Glucocorticoid hormones have dramatic effects on cytodifferentiation and carcinogenesis in vitro and in vivo. We have investigated the effects of the endogenous rodent glucocorticoid hormone, corticosterone, on the X-irradiation-induced transformation and differentiation of C3H/10T1/2 mouse fibroblast cells in culture. Initially, we have demonstrated the presence of functional glucocorticoid receptors in these cells. We found that corticosterone has little effect on X-ray-induced transformation. However, this hormone alone was found to differentiate a high number of these fibroblastic cells to the adipogenic cell lineage. Using an antagonist to the glucocorticoid receptor, we demonstrate that the hormonal effect on differentiation is mediated by the corticosterone-receptor complex.

Animals↗
PubMed1988

Mesodermal cell lineage determination and proto-oncogene expression.

Previously, we have established a rat embryo fibroblastic cell line that is characterized as immortal, nontumorigenic and retains a diploid karyotype. We report here that this cell line will spontaneously differentiate along mesodermal cell lineages to form myotubes and adipocytes. We have isolated and characterized lineage-determined (nondifferentiated) preadipocytes and myoblasts. Using these cell lines we have investigated the expression of proto-oncogenes concomitant with lineage-specific determination. No major changes in proto-oncogene RNA levels were observed that correlated with lineage determination. These results would suggest that none of the 15 proto-oncogenes used in these experiments are involved in mesodermal lineage determination; but this does not rule out the possibility that other proto-oncogenes not tested or currently unknown may be involved. However, these differentiating subclones and nondifferentiating fibroblasts that exhibit a normal karyotype will be an excellent model-system to investigate the molecular basis of cell lineage determination.

Adipose Tissue↗
PubMed1988

Escape from X-ray-induced arrest for lens cells stimulated from quiescence: time relationship to RNA, protein, and DNA synthesis.

Quiescent cells of the central zone region of the rat lens epithelium were stimulated to enter the proliferation cycle by wounding. RNA synthesis and a corresponding increase in poly(A)+/total RNA reached a peak by Hour 4. Cells progressed into the G1B compartment by Hour 10. A rise in protein synthesis began at Hour 8, and onset of DNA synthesis occurred by Hour 14. The timing of cell cycle progression that allowed escape from a dose of X irradiation that completely inhibited DNA synthesis was investigated. A growth-arrest point was identified at Hour 9 where 10 GY of X irradiation given before, but not after, completely inhibited earliest responding cells from entering DNA synthesis on schedule. Increased quantities of cells entered DNA synthesis on schedule as timing of the X irradiation was moved closer to the end of G1. Based on time relationships, the rise in protein synthesis is correlated with the "sufficient" event for the escape.

Animals↗
PubMed1987

Correlation of thyroid hormone dose-dependent regulation of K-ras protooncogene expression with oncogene activation by 3-methylcholanthrene: loss of thyroidal regulation in the transformed mouse cell.

Previously, it has been demonstrated that thyroid hormone is an important cofactor of the initiation of oncogenesis in vivo and in vitro. In order to determine the mechanism of thyroid hormone modulation of the initiation of carcinogenesis we have addressed the hypothesis that thyroid hormone regulates the expression of the critical protooncogene at the time of exposure to the carcinogen, and that the transcriptional activity of the protooncogene correlates with the ability of a carcinogen to "activate" the oncogene and thus modulate the subsequent transformation event. It has previously been shown that 3-methylcholanthrene transformation of C3H/10T1/2 mouse embryo cells in culture is the result of activation of the k-ras oncogene. We report here that thyroid hormone modulates 3-methylcholanthrene transformation of C3H/10T1/2 cells in a dose-dependent manner that is similar to a thyroid hormone dose-dependent modulation of k-ras-specific RNA levels in these cells. Further, nuclear transcriptional run-on experiments suggest that the thyroidal-induced changes in K-ras RNA levels are a result of a regulation of K-ras transcription. These data support the hypothesis that thyroid hormone modulation of transformation is through regulation of protooncogene expression. It was of further interest to find that 3-methylcholanthrene-transformed C3H/10T1/2 cells have lost the sensitivity to thyroid hormone regulation of "activated" K-ras oncogene transcription and subsequent K-ras-specific RNA levels.

Animals↗
PubMed1987

Regulation of thyroidal inducibility of Na,K-ATPase and binding of epidermal growth factor in wild-type and cold-sensitive E1a mutant type 5 adenovirus-transformed CREF cells.

We have analyzed the relationship between expression of the transformed phenotype and thyroid hormone (triiodothyronine, T3) inducibility of Na,K-ATPase and binding of 125I-epidermal growth factor (EGF) to cell membrane receptors in wild-type (wt) and mutant type 5 adenovirus (Ad5)-transformed CREF cells displaying a cold-sensitive (cs) expression of the transformed phenotype. CREF cells respond to thyroid hormone treatment with increased Na,K-ATPase activity and bind similar levels of 125I-EGF at 32 degrees C, 37 degrees C and 39.5 degrees C. In contrast, CREF cells transformed by wt Ad5 or the E1a plus E1b-transforming genes of wt Ad5 are refractile to T3 treatment and bind lower levels of 125I-EGF than CREF cells at all three temperatures. By employing a series of cloned CREF cell lines transformed by a host-range cold-sensitive mutant virus, H5hr1 or H5dl101, or the E1a or E1a plus E1b genes from these viruses, we have investigated expression of the transformed state and its relationship with hormone inducibility and EGF binding. When cs virus, cs E1a- or cs E1a plus E1b-transformed CREF clones were grown at 32 degrees C, a nonpermissive transforming temperature in which cs-transformed cells exhibit properties similar to untransformed CREF cells, T3 induced Na,K-ATPase activity and these cells bound similar levels of 125I-EGF as CREF cells. However, when cs virus- and cs Ela plus E1b-transformed CREF clones were incubated at 37 degrees C or 39.5 degrees C, temperatures at which cs-transformed cells exhibit properties similar to wt Ad5-transformed CREF cells, they did not respond to T3 and bound lower levels of 125I-EGF than CREF cells. In the case of cs E1a-transformed CREF clones, thyroid hormone responsiveness was observed at both 32 degrees C and 37 degrees C, but not at 39.5 degrees C. By performing temperature shift experiments--i.e. 32 degrees C to 37 degrees C, 32 degrees C to 39.5 degrees C, 37 degrees C to 32 degrees C, and 39.5 degrees C to 32 degrees C, it was demonstrated that after a shift from lower to higher temperature a 24-hr lag period was required for cs-transformed CREF cells to lose T3 inducibility and exhibit reduced EGF binding, whereas 96 hr after a shift from higher to lower temperature a 96-hr lag period was required for cs-transformed cells to regain T3 inducibility and increased 125I-EGF binding.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenoviridae↗
PubMed1987

Thyroid hormone affects the expression of neoplastic transformation induced by DNA-transfection.

Thyroid hormone can dramatically modulate oncogenic transformation of cells in culture. To further investigate this we have used DNA-mediated gene transfer (transfection) to transform cells grown in the presence (+T3) or absence (-T3) of thyroid hormones. Removal of thyroid hormones from the culture media greatly reduced the appearance of transformed foci subsequent to transfection. However, +T3 or -T3 media had no effect on the appearance of ouabain-resistant (ouar) colonies following transfection of ouabain-sensitive (ouas) cells with DNA isolated from ouar cells and selection in 3 mM ouabain. These results suggest that thyroid hormone does not effect the uptake or integration of exogenous DNA, but instead may modify the expression of transformation.

Animals↗
PubMed1986

Loss of thyroidal inducibility of Na,K-ATPase with neoplastic transformation in tissue culture.

Thyroidal induction of the plasma membrane Na,K-ATPase is a characteristic of mammalian tissues that exhibit a thermogenic response to this hormone. To facilitate analysis of the pathways mediating this response, we defined the conditions needed for reproducible thyroidal induction of this enzyme, as well as mitochondrial cytochrome c oxidase, in established cell lines in tissue cultures. In confluent monolayers of nontransformed mouse embryo fibroblasts (C3H/10T1/2), triiodothyronine modulated Na,K-ATPase and cytochrome c oxidase activities in a concentration- and time-dependent manner. Similar increases in Na,K-ATPase activity were obtained in other rodent embryo cells (SWISS/3T3 and NIH/3T3) and in human fibroblasts (WI-38). In contrast, neoplastic transformation of all of these cell lines resulted in loss of inducibility of Na,K-ATPase by thyroid hormone, regardless of the initiating mechanism (i.e. spontaneous, x-ray, chemicals, viruses).

5'-Nucleotidase↗
PubMed1986

An altered response in the induction of cell membrane (Na + K)ATPase by thyroid hormone is characteristic of senescence in cultured human fibroblasts.

There have been numerous investigations of thyroid function during senescence in humans. However, very little information is available on thyroid hormone action at the cell level during senescence. Therefore, we have investigated thyroid hormone induction of cell membrane (Na + K)ATPase during human senescence using three experimental fibroblast cell culture systems: (1) cells from premature aging syndrome, progeria; (2) aging in vitro; and (3) early passage cells from aged patients. In all cases senescence is associated with a dramatic alteration from the normal dose-dependent thyroid hormone induction of (Na + K)ATPase. Senescent cells depleted of thyroid hormones demonstrated an elevated activity of (Na + K)ATPase, while non-senescing cells exhibit the characteristic basal enzyme activities in the hypothyroid state. These results indicate that human senescence is associated with extreme alterations in thyroid hormone regulation of (Na + K)ATPase; and may suggest a more general change in thyroid hormone action at senescence. These changes may be associated with important alterations in cell metabolism and intracellular ionic environment during senescence.

Adolescent↗