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Biomedical subjects

T H Norwood

Publications and source records attributed to T H Norwood.

At least 19 recordsLinked to original sources
PubMed2004

A distinctive nuclear morphology in acute myeloid leukemia is strongly associated with loss of HLA-DR expression and FLT3 internal tandem duplication.

In a 5-year survey of nonpromyelocytic/nonmonocytic acute myeloid leukemias (AMLs) diagnosed in the University of Washington Hematopathology Laboratory, we identified 19 cases containing distinctive, cup-like nuclear indentation in 10% or more of the blasts ('AML-cuplike'). Fourteen of these cases (74%) demonstrated near-complete loss of HLA-DR expression, while the other five cases showed partial loss of HLA-DR. A total of 16 of the cases (84%) demonstrated internal tandem duplication (ITD) of the Flt3 gene. When compared to a selected set of AMLs lacking this nuclear morphology, AML-cuplike was significantly more likely to lack HLA-DR and CD34 expression, to express CD123 without CD133, to have a normal karyotype, and to harbor the Flt3 ITD. To characterize AML-cuplike in an unselected series of AMLs, we analyzed 42 consecutive nonpromyelocytic/nonmonocytic AMLs diagnosed in our laboratory during a 6-month period in 2002. Strikingly, in this unselected series, there was a statistically significant coincidence of invaginated nuclear morphology, loss of HLA-DR, and presence of the Flt3 ITD beyond that expected if these three features were unrelated, suggesting that AMLs with these three features may represent a distinct AML subset.

Acute Disease↗
PubMed2001

Chromosomal changes in a dedifferentiated chondrosarcoma: a case report and review of the literature.

The chromosome abnormalities observed in a dedifferentiated chondrosarcoma are reported. A new molecular cytogenetic technique, spectral karyotyping, was used to identify and confirm structural rearrangements in this case. A review of the literature revealed that nine cases have been reported, in eight of which a complete description of the cytogenetic abnormalities was described. Structural aberrations were most frequently reported in chromosomes 1 and 9, and chromosomes 7 and 19 were most frequently observed to be involved in numerical aberrations (trisomy and tetrasomy). In chondrosarcomas, structural aberrations in chromosomes 1 and 9 and trisomy or tetrasomy of chromosome 7 are among the more frequently observed aberrations.

Arthroplasty, Replacement, Knee↗
PubMed2000

Chimeric del20q in a case of chronic neutrophilic leukemia.

Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative disorder in which recurrent abnormalities of chromosome 20 have been reported. We report the case of a 76-year-old woman with CNL with partial deletion of the long arm of chromosome 20 in a subset of bone marrow metaphases, suggesting coexistence of a clonal stem cell disorder and normal hematopoiesis. Review of the literature suggests that such mosaicism is common in CNL, possibly accounting for the favorable prognosis observed in many patients with this disorder.

Aged↗
PubMed1999

A novel human gene encoding HECT domain and RCC1-like repeats interacts with cyclins and is potentially regulated by the tumor suppressor proteins.

Cyclin E-Cdk2 is an evolutionary conserved cyclin-dependent kinase (CDK) complex that drives the G1 to S phase transition of the cell cycle. A novel cDNA encoding a HECT family protein also containing RCC1-like repeats was isolated by a yeast two-hybrid screening using both cyclin E and its inhibitor p21. The protein product of this cDNA, Ceb1, interacts with various cyclin subunits of CDKs in mammalian cells. Expression of Ceb1 is specifically detected in testis and ovary and is highly elevated when the functions of the tumor suppressor proteins, p53 and RB, are compromised by mutations or viral oncoproteins. The present results suggest that Ceb1 may play a critical role when its expression and the CDK activity are upregulated by inactivation of p53 and RB.

Amino Acid Motifs↗
PubMed1999

Analysis of the capacity of extracts from normal human young and senescent fibroblasts to support DNA synthesis in vitro.

Cytoplasmic extracts from early-passage (young), late-passage (senescent) normal human fibroblast (HF) cultures and immortalized human cell lines (HeLa, HT-1080, and MANCA) were analyzed for their ability to support semiconservative DNA synthesis in an in vitro SV40-ori DNA replication system. Unsupplemented extracts from the three permanent cell lines were demonstrated to be active in this system; whereas young HF extracts were observed to be minimally active, and no activity could be detected in the senescent HF extracts. The activity of these extracts was compared after supplementation with three recombinant human replication factors: (1) the catalytic subunit of DNA polymerase alpha (DNA pol-alpha-cat), (2) the three subunits of replication protein A (RPA), and (3) DNA topoisomerase I (Topo I). The addition of all three recombinant proteins is required for optimum activity in the young and senescent HF extracts; the order of the level of activity is: transformed > young HF > senescent HF. Young HF extracts supplemented with RPA alone are able to support significant replicative activity but not senescent extracts which require both RPA and DNA pol-alpha-cat for any detectable activity. The necessary requirement for these factors is confirmed by the failure of unsupplemented young and senescent extracts to activate MANCA extracts that have been immunodepleted of DNA pol-alpha-cat or RPA. Immunocytochemical studies revealed that RPA, DNA pol-alpha, PCNA, and topo I levels are higher in the immortal cell types used in these studies. In the HF cells, levels of DNA pol-alpha-cat and PCNA are higher (per mg protein) in the low-passage than in the senescent cells. By contrast, RPA levels, as determined by immunocytochemical or Western blot studies, were observed to be similar in both young and senescent cell nuclei. Taken together, these results indicate that the low to undetectable activity of young HF extracts in this system is due mainly to reduced intracellular levels of RPA, while the senescent HF extracts are relatively deficient in DNA polymerase alpha and probably some other essential replication factors, as well as RPA. Moreover, the retention of RPA in the senescent HF nuclei contributes to the low level of this factor in the cytoplasmic extracts from these cells.

Blotting, Western↗
PubMed1998

Role of cyclin E and cyclin E-dependent kinase in mitogenic stimulation by cementum-derived growth factor in human fibroblasts.

Cementum-derived growth factor (CGF) is a 14 kDa polypeptide sequestered in tooth cementum. It is an IGF-I like molecule that is weakly mitogenic to fibroblasts, but its mitogenic action is synergistically potentiated in the presence of epidermal growth factor (EGF) or serum. We have examined whether the CGF affects cyclin E levels and the activity of cyclin-dependent kinase (Cdk) associated with this cyclin, and whether these changes contribute to the synergism in mitogenic activity between CGF and EGF. Optimal DNA synthesis by serum-starved human gingival fibroblasts required the presence of CGF for 0-12 h and EGF for 0-3 h. Therefore, cells were serum starved for 48 h and then exposed to CGF, EGF, or CGF + EGF. Cells incubated with 10% fetal bovine serum (FBS) served as positive controls. At various time points after the addition of growth factors, cyclin E levels were examined by Western analysis. Cdk associated with cyclin E was immunoprecipitated with anti-cyclin E antibody and kinase activity was measured using H1 histone as substrate. Cyclin E and the H1 kinase activity levels increased after 8-12 h in cells exposed to CGF and in positive controls exposed to 10% FBS. They returned to basal level 4 h later in cells exposed to CGF alone, whereas in the presence of CGF + EGF and FBS they remained elevated for up to 20 h. The cyclin E levels did not increase in the presence of EGF alone. Cyclin-dependent kinase inhibitors p21cip1 and p27kip1 were barely detectable in these cells. Fibroblasts transfected with LXSN-cyclin E, a retroviral vector containing cyclin E cDNA, overexpressed cyclin E and their steady-state cyclin E-Cdk activity was higher than control cells. DNA synthesis by cyclin E overexpressing cells was higher, but optimal DNA synthesis by these cells required the presence of CGF and EGF. These results show that CGF action involves an increase in the levels of cyclin E and E-Cdk activity and that the higher levels are maintained in the presence of both CGF and EGF. They also indicate that sustained high cyclin E levels and Cdk2 activity during G1 phase are necessary, but not sufficient, for optimal mitogenic response in human fibroblasts.

Animals↗
PubMed1996

Trisomy 10p: report of an unusual mechanism of formation and critical evaluation of the clinical phenotype.

A de novo tandem inverted duplication of 10p was diagnosed in a 17-week fetus. The appearance of GTG banded preparations and the results of fluorescence in situ hybridization (FISH) studies are consistent with duplication of the entire arm, including the telomere. The FISH studies also demonstrated the presence of chromosome 10 alphoid repeats at the junction between the inverted segment and the long arm, consistent with the presence of the entire long arm of the abnormal chromosome. Therefore, this is a case of pure trisomy 10p without an associated deficiency of any other chromosome segment. A comparison of the phenotype associated with pure trisomy 10p and trisomy associated with a duplication/deficiency state documented a higher frequency (of borderline significance) of clubfoot and high-arched/cleft palate in the cases of pure trisomy. The frequency of palatal anomalies was observed to be significantly higher in the cases where the breakpoint of the trisomic segment is in the most proximal band (10p11). However, other clinical manifestations were observed inconsistently, even in the cases with pure, nearly complete trisomy 10p. Therefore, a clearly defined trisomy 10p clinical syndrome could not be documented in this study.

Adult↗
PubMed1995

Balanced reciprocal translocation mosaicism: how frequent?

We describe 2 cases of balanced reciprocal translocation (BRT) mosaicism. The frequency of this aberration in the population referred to our laboratory was determined and compared to those frequencies reported in the literature by other clinical cytogenetics laboratories. The extent of BRT mosaicism was also examined in surveys of parental populations, which are less likely to have a bias due to ascertainment on the basis of abnormal phenotype. the frequencies in the postnatal and prenatal populations examined in this study were calculated to be 5.7 x 10(-5) (95% confidence interval is 3.2-8.2 x 10(-5)) and 4.1 x 10(-5) (95% confidence interval is 2.0-6.2 x 10(-5)). However, in view of the extent of variation reported in the various studies, these estimates should be considered first approximations of the true frequency.

Abnormalities, Multiple↗
PubMed1994

Murine temperature-sensitive DNA polymerase alpha mutant displays a diminished capacity to stimulate DNA synthesis in senescent human fibroblast nuclei in heterokaryons at the nonpermissive condition.

We have investigated the capacity of a murine cell line with a temperature-sensitive (ts) mutation in the DNA polymerase alpha (Pola) locus and a series of ts non-Pola mutant cell lines from separate complementation groups to stimulate DNA synthesis, in senescent fibroblast nuclei in heterokaryons. In the Pola mutant x senescent heterodikaryons, both human and murine nuclei display significantly diminished levels of DNA synthesis at the restrictive temperature (39.5 degrees C) as determined by [3H]thymidine labeling in autoradiographs. In contrast, all of the non-Pola mutants, as well as the parental (wild type) murine cells, induced similar levels of DNA synthesis in both parental nuclei at the nonpermissive and permissive temperatures. Similarly, young human fibroblasts are also able to initiate DNA synthesis in heterokaryons with the ts Pola mutant at the two temperatures. In order to determine if complementation of the non-Pola mutants requires induction of serum responsive factors in the senescent cells, fusion studies of similar design were conducted with young and old human fibroblasts incubated in low serum (0.2%) for 48 hr prior to and after cell fusion. Again, a diminished level of DNA synthesis was observed at 39.5 degrees C in the Pola mutant x senescent cell heterokaryons. In these low-serum studies, both parental nuclei in the Pola x young cell heterokaryons and the human nuclei in heterokaryons with one of the non-Pola mutants (FT107) also displayed diminished levels of DNA synthetic activity. All of the other mutants are able to support similar levels of synthetic activity at both temperatures in the presence of reduced serum. The nature of the mutation in three of the non-Pola lines has not been determined but, like the Pola mutant cells, are inhibited in the G1 phase of the cell cycle when incubated at the nonpermissive temperature (39.5 degrees C). The fourth non-Pola mutant line is known to have at least one ts mutation in the cdc2 gene and is inhibited in the G2 phase when exposed to 39.5 degrees C. These results suggest that there may be a functional deficiency of pol alpha in senescent human fibroblasts, and this replication factor may be one of the rate-limiting factors involved in loss of the capacity to initiate DNA synthesis in senescent cells.

Animals↗
PubMed1993

Mosaic isochromosome 8p.

Findings in a patient with mosaic isochromosome 8p are compared with those of previously reported cases. There were no distinguishing findings on physical examination; all had cognitive delays, especially in speech and language development.

Abnormalities, Multiple↗
PubMed1993

A simple method of rapid freezing adequately preserves brain tissue for immunocytochemistry, light and electron microscopic examination.

A simple and reproducible method for cryopreservation of brain tissue from patients with Alzheimer's disease is described. Fresh brain slices (1 cm thick) obtained less than 6 h postmortem are placed in sealed plastic bags, sandwiched between 0.3-cm-thick aluminium sheets, and frozen by placing the entire "sandwich" between layers of dry ice pellets. The frozen brain slices are stored at -85 degrees C. Specific anatomic areas can be retrieved at any time for light and electron microscopic, immunocytochemical, autoradiographic and neurochemical studies.

Aged↗
PubMed1992

The cultured diploid fibroblast as a model for the study of cellular aging.

The limited proliferative potential of the cultured human diploid fibroblast is now well established. A number of biological correlates suggest that this culture system is a model for the study of aging at the cellular level. The mechanism(s) that causes the loss of proliferative activity is unknown; the results of some recent studies indicate that specific genes may play a pivotal role in cellular aging in vitro. The extent to which changes in proliferative functions are causally related to aging in vivo is currently under investigation.

Cells, Cultured↗
PubMed1991

Mutants from V79 fibroblasts exhibiting hypersensitivity to aphidicolin and 3'-azido-3'-deoxythymidine.

One variant, aphhs-3 was previously isolated based on a hypersensitivity to nontoxic concentrations of aphidicolin, a specific inhibitor of DNA polymerases-alpha and delta. This variant was found to be more sensitive to temperatures above 35 degrees C and to 10 microM of 3'-azido-3'-deoxythymidine (zidovudine, azidothymidine, or AZT) than the parental 743x cells. DNA polymerase activities in the cell extract or in the partially purified fraction by DEAE-cellulose (DE52) anion exchange column from aphhs-3 were active at 40 degrees C. No significant differences in deoxynucleoside triphosphate pools were observed at 34 degrees C for both the parental 743x and aphhs-3 cells. Revertants were isolated at 39 degrees C: six revertants (aphhs-3-tr1 through aphhs-3-tr6) were obtained without aphidicolin; one revertant aphhs-3-tar (the tar clone) was selected in aphidicolin (0.12 microM). The hypersensitivity to aphidicolin (Aphhs) and AZT (AZThs) was cosegregated in the revertant aphhs-3-tr5 (the tr5 clone), while the tar clone was not AZThs. There was a similar increase in the specific activity of 3H-labeled DNA in all cell lines after additions of [3H]AZT or [3H]thymidine. Additions of purine or pyrimidine arabinosides (araT, araC, and araA) to all cell lines resulted in a similar cytotoxicity, suggesting the anabolism of dTTP was not defective in the tr5 clone. The spontaneous mutation rate at the hypoxanthine-guanine phosphoryltransferase locus using replating techniques and 6-thioguanine resistance selection was less than or equal to 5 x 10(-7), 2.2 x 10(-6), or 1.3 x 10(-6) per generation for the tr5, 743x, or tar cell lines, respectively. Most importantly, DNA polymerase activities in the cell extract of the revertant tr5 clone were inhibited by 0.5 microM AZTTP. In contrast, no inhibition was observed in those of the parental 743x and revertant tar cells. The cosegregation of both Aphhs and AZThs in the tr5 revertant suggests that these two phenotypes may be a result of the same mutational event.

Animals↗
PubMed1991

The relationship between the rate of entry into S phase, concentration of DNA polymerase alpha, and cell volume in human diploid fibroblast-like monokaryon cells.

We have examined the kinetic relationship between the rate of entry into the S phase in human diploid fibroblast-like (HDFL) monokaryon cells and (1) the concentration of DNA polymerase alpha activity and (2) the cell volume. In the former studies, a first-order dependence between the rate of entry into the S phase and the concentration of DNA polymerase alpha activity was observed, consistent with the enzyme, or a coregulated factor, being rate limiting for this metabolic process. Examination of the nature of the dependence of the rate of entry into the S phase upon cell volume revealed a more complex relationship. The results obtained in studies with synchronized cultures are consistent with the presence of two to three rate-limiting reactants when cell volume is the independent variable. Studies with asynchronous HDFL cell cultures revealed that the smallest cells in the G1 population, presumably the early G1 cells, enter the S phase at an increasing rate as a function of cell volume up to a certain size, beyond which the cells enter at a decreasing rate similar to that observed in the studies with the synchronized cultures. Similar studies examining the relationship between cell volume and the rate of entry into S phase in three established immortal cell lines revealed positive correlation between the rate of entry into S phase and cell volume throughout the size range of the G1 population. This latter observation suggests that the factors involved in the initiation of the S phase may be present in concentrations that are not rate limiting in immortal cell lines.

Cell Division↗
PubMed1991

DNA polymerase alpha and the regulation of entry into S phase in heterokaryons.

We have previously reported that the DNA polymerase alpha activity/unit cellular protein is decreased in late-passage (senescent) human diploid fibroblast-like (HDFL) cultures due to the cellular enlargement associated with in vitro aging. In the studies described here, we have used cell fusion technology to investigate the formal kinetic relationship between the concentration of DNA polymerase alpha and the rate of reinitiation of DNA synthesis in nuclei from senescent cells. Heterokaryons were derived from the fusion of senescent cells to a series of actively dividing cell types with inherently different DNA polymerase alpha activities per cell. A kinetic analysis revealed a first-order relationship between the entry into S phase of senescent nuclei and the concentration of DNA polymerase alpha activity calculated to be in heterokaryons. This result suggests that increases in cell volume may be related to the decline in proliferative activity of late-passage HDFL cells, via "dilution" of factors essential for cellular replication.

Cell Division↗
PubMed1989

Cell enlargement: one possible mechanism underlying cellular senescence.

We previously demonstrated an inverse relationship between the G1 volume of human diploid fibroblast-like (HDFL) cells obtained from foreskin tissue and clonal replicative potential. On the basis of these results, we suggested that one process underlying in vitro senescence is a progressive increase in the mean cell volume of successive progeny within clonal lineages. We now report that the size of HDFL cells, as well as of chick embryo fibroblasts, can be increased in the virtual absence of cell division by culturing at low density and at low serum concentration (0.1-1.0%). Consequent to an increase in cell size, the replicative potential of the cells is reduced to the level of later-passage cells of similar size. By clonal analysis, the populations of enlarged cells contain up to three times as many nondividing cells as do controls. In the enlarged populations, the proportion of cells producing attenuated clones (four or fewer progeny) increases by about 30%, whereas the proportion of cells yielding greater than 32 cells declines by a similar percentage. These observations lead us to propose that replicative potential may be limited by cell size, which in turn may be regulated by a kinetic relationship between cellular growth and cell division cycles.

Animals↗
PubMed1989

The relationship between cell size, the activity of DNA polymerase alpha and proliferative activity in human diploid fibroblast-like cell cultures.

In kinetic studies with human diploid fibroblast-like (HDFL) cells carried out in heterokaryons and in monokaryons, we have observed a first-order relationship between the level or concentration of DNA polymerase alpha and the rate of initiation of new rounds of DNA synthesis. Because cell size is inversely proportional to the concentration of DNA polymerase alpha and presumably other replication factors, it is inversely related to the initiation of new rounds of DNA synthesis. An inverse relationship between cell size and clonogenic activity was also observed in both unsorted HDFL cells and in HDFL cells sorted on the basis of size. Experimental enlargement of cells by serum deprivation at low density resulted in changes in colony-forming ability that would be predicted by these studies. A causal relationship between the observed increase in cell size with advancing passage level and the loss of proliferative activity is suggested by these studies; in addition, cell size may be a useful biophysical marker for cellular aging.

Cell Division↗