Biomedical subjects
J W Freeman
Publications and source records attributed to J W Freeman.
On-call perspective.
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Dealing with devastation.
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Altered transcription control is responsible for the increased level of proliferation-associated P120 in rapidly growing breast carcinoma.
Transcriptional and post-transcriptional regulation of proliferation-associated nucleolar P120 protein expression was examined in 1 normal and 5 malignant breast cell lines. The 6 breast cell lines could be placed into 3 categories on the basis of in vitro growth rate. BT549 and HBL100 grew rapidly; MCF-7/6, MCF-7/AZ and Hs578T grew at a moderate rate; and Hs578N normal epithelia grew slowly. There was a significant correlation between the growth rate, measured by percentage of S-phase fraction of cells or doubling time of cell numbers and the steady-state levels of either P120 protein or P120 mRNA. Next, the mechanisms responsible for the increased level of P120 expression, which is associated with rapidly growing breast carcinoma, were examined. The stability of P120 mRNA was measured by densitometry of quantitative Northern blots of mRNAs from cells treated with actinomycin D. Before the expected decay, a sharp increase in P120 mRNA level was detected shortly after inhibiting the overall RNA or protein synthesis. The calculated half-life of P120 mRNA was very similar (1.8 +/- 0.2 hr) in all 6 cell lines examined. The transcription rate of the P120 gene was determined by densitometric analysis of quantitative nuclear run-off assays. A significant positive correlation was found between the transcription rate of the P120 gene and the steady-state levels of either P120 protein or P120 mRNA. Our conclusion is that the expression of P120 protein is transcriptionally regulated in these breast cells. Therefore, the characteristically high level of P120 protein and mRNA found in most breast carcinomas is due to the altered transcription rate of the P120 gene in transformed cells.
Nucleolar p120 is expressed as a delayed early response gene and is inducible by DNA-damaging agents.
Regulation of the expression of the growth-related nucleolar p120 protein was examined in serum-deprived and stimulated nontransformed and SV40-transformed WI-38 human fibroblasts. In quiescent cells, transcriptional activity of the p120 gene was very low or undetectable, and the steady-state levels of the p120 mRNA and the p120 protein were also negligible. The transient expression of the p120 gene in the cell cycle was detected in middle G1-phase after the expression of the early response genes and before the expression of the DNA-synthesis genes. Protein synthesis was required for the induction of p120 expression in serum-stimulated cells. The increased level of p120 mRNA in middle G1-phase was attributed to an increased transcription rate of the p120 gene, and not to a change in p120 mRNA stability. The calculated half-life of p120 mRNA was unchanged (1.8 +/- 0.2 hr) in all four cell conditions tested; i.e., in middle G1- or S-phase cells and in exponentially growing normal or transformed cells. Transcription rate of the p120 gene was correlated with the steady-state levels of either p120 protein or p120 mRNA. A sharp increase in p120 mRNA level occurred in both normal and transformed cells treated with actinomycin D used to examine p120 mRNA stability. This induction of p120 mRNA expression was seen in early G1-phase, but not in quiescent cells, or in middle to late G1-phase when cells expressed the highest level of p120 mRNA. The same expression pattern was seen by treatment with chlorambucil, another DNA-damaging agent. The conclusions of these studies are that the expression of p120 (1) is serum inducible in a fashion characteristic of the delayed early response gene products, (2) requires the presence of newly synthesized proteins, (3) is regulated transcriptionally, and (4) can be induced by DNA-damaging agents.
Increased tumorigenicity in the human pancreatic cell line MIA PaCa-2 is associated with an aberrant regulation of an IGF-1 autocrine loop and lack of expression of the TGF-beta type RII receptor.
The growth characteristics associated with tumorigenicity were determined in clones of MIA PaCa-2 and PANC-1 pancreatic carcinoma cells. MIA PaCa-2 cells differed from PANC-1 cells in that they rapidly formed tumors in nude mice, formed colonies more rapidly and formed larger colonies in soft agar, and were cloned more efficiently when seeded at low density. MIA PaCa-2 cells but not PANC-1 cells were stimulated to escape quiescence and undergo DNA synthesis with nutrient media lacking growth factors. Both cell lines were stimulated to proliferate with serum-free media containing EGF, transferrin, and insulin. Antibody neutralization assays indicated that an IGF-1 autocrine loop was required for the nutrient stimulation of growth in MIA PaCa-2 cells and for the growth-factor stimulation in both MIA PaCa-2 and PANC-1 cells. Both cell lines were stimulated to proliferate with exogenous IGF-1 in basal media; this stimulation was specifically blocked by antibodies to IGF-1 or its receptor. MIA PaCa-2 and PANC-1 cells expressed similar levels of IGF-1 receptor mRNA and showed similar binding kinetics in receptor binding assays. In contrast to PANC-1 cells, MIA PaCa-2 cells were insensitive to TGF-beta 1 and did not express TGF-beta receptor type II. The results suggest that the growth-factor independence is representative of a more tumorigenic phenotype. We hypothesize that growth-factor independence of MIA PaCa-2 cells is mediated by an aberrant regulation of an IGF-1 autocrine loop. A decreased regulation of this IGF-1 loop may be potentiated by loss of response to TGF-beta.
Prevention of hypothermia during orthotopic liver transplantation: comparison of three different intraoperative warming methods.
Hypothermia is a frequent and sometimes clinically important problem during orthotopic liver transplantation. Numerous methods have been suggested to reduce intraoperative heat loss and promote active warming. In this study we compared an electric under mattress, a warm air under mattress and a forced warm air convective heating blanket. The forced air convective warming system was shown to produce significantly higher patient temperatures than the two other systems.
Traumatic abducens nerve paresis in a child.
Trauma is a frequent cause for abducens (sixth) nerve paresis in a child, usually attributed to injury along the nerve's course. An unusual focal lesion of the sixth nerve nucleus is described.
Caring and the health professions.
This study analyzes patient descriptions of what constitutes caring in the physician and nurse. We propose that health care students can be taught caring behavior through the use of patient narratives.
Expression of proliferation-associated antigens (PCNA, p120, p145) during the reentry of G0 cells into the cell cycle.
Flow cytometric bivariate analysis was used to evaluate the expression of PCNA, p120, and p145 during the G0 reentry of CHO-K1 cells into the cell cycle. CHO-K1 cells were placed in a G0-like state using serum depletion and stimulated to reenter the cell cycle by replating into fresh, serum-containing medium. At discrete intervals after stimulation, replicate samples were stained for either PCNA, p120, or 145; stained for DNA (Coulter DNA-Prep); evaluated on the EPICS Profile I; and analyzed on the EPICS ELITE workstation. PCNA stained less than 10% of the G0 cells; in contrast, however, 30-35% of the G0 cells were positive for p120 and p145. Eight hours after stimulating G0 cells to reenter the cell cycle (during G0/G1), p120 reached 88% positivity, while p145 and PCNA were 63% and 30% positive, respectively. Cells in S phase (12 and 16 h following G0 stimulation) were greater than 90% positive for all three antigens. PCNA had the greatest change throughout the G0 reentry process, both in percentage positive and quantitatively (mean channel fluorescence). This report indicates that all three proliferation-associated antigens studied are differentially expressed during the reentry of G0 cells into the cell cycle. Furthermore, these antigens may be useful in the early detection of G0 recruitment.
Effect of nucleolar P120 expression level on the proliferation capacity of breast cancer cells.
Steady-state level of nucleolar P120 protein and P120 mRNA was compared to the doubling time and S-phase fraction in human breast cancer cell lines growing exponentially and in similar cells treated with a single dose of P120 antisense oligodeoxynucleotides. The study included six breast cancer cell lines and one nontransformed breast cell line with doubling times from 1.1 to 5.5 days and with S-phase fractions from 35 to 9%. P120 expression level was determined by densitometric computerized evaluation of protein and mRNA blots and with a quantitative 32P-reverse transcriptase-polymerase chain reaction method developed for small-scale samples. In the slowest growing normal cell line, P120 expression level was only about 10% of the level found in the most rapidly growing cancer cell line. The amount of P120 mRNA was highly correlated with the amount of P120 protein (P = 0.0001), indicating that P120 accumulation is regulated in these cells primarily at a transcriptional level. There was also a significant positive correlation between the level of P120 protein/mRNA and doubling time of cell lines (P = 0.0008) or percentage of S-phase cells (P = 0.210). P120 antisense oligomer treatment decreased the growth rate of cells in a dose-dependent manner, and the inhibition reached 70% at 100 microM concentration. Both P120 mRNA and P120 protein levels were also decreased by approximately 70% in cells treated with 100 microM P120 antisense oligomer. Slowly growing cells exhibited 50% inhibition by treatment at a proportionally lower concentration of P120 antisense oligomer than fast growing cells. This study shows that the expression of P120, measured either at the protein or the mRNA level, correlates with proliferation rate, identifying P120 as a cell proliferation marker.
Improved survival with adjuvant immunotherapy after surgical resection in a murine model.
BACKGROUND: Adoptive immunotherapy has met with limited success in the treatment of bulky metastatic disease. The purpose of this study was to determine whether lymphocytes stimulated in vitro could improve survival when given as an adjuvant to surgical resection in animals harboring microscopic metastatic disease. METHODS: Lymphocytes from nodes draining the primary tumor (DLN lymphocytes) were stimulated in vitro with phorbol 12,13-dibutyrate and ionomycin and used as adjuvant immunotherapy after surgical resection of the primary tumor. Mice with advanced P-815 footpad tumors and disseminated microscopic metastases underwent amputation of the tumor-bearing extremity and were randomized to various adjuvant treatments. RESULTS: Mice treated with adjuvant immunotherapy using stimulated DLN lymphocytes demonstrated significantly improved survival, showing that DLN lymphocytes stimulated in vitro can abrogate metastases that are invading multiple organs simultaneously. Mice successfully treated with adjuvant immunotherapy demonstrated long-term (80 days) in vivo antitumor activity by rejecting subsequent tumor challenge. In addition, stimulated DLN lymphocytes provided in vivo antitumor activity to naive mice. CONCLUSIONS: Adjuvant immunotherapy after resection in the face of residual microscopic tumor burden may prove to be a useful application of adoptive immunotherapy.
Prospective study correlating P120 antigen expression with established prognostic factors in breast cancer.
P120 is a nucleolar proliferation antigen found in rapidly dividing cells and a variety of malignancies. Previous retrospective studies have demonstrated that, when detected in human breast cancer, P120 is associated with a poorer prognosis. To determine whether P120 expression correlates with other prognostic factors in breast cancer, we prospectively analysed pathologic and clinical data from 61 patients. P120 was detected in 40 of the 61 specimens (66%). No significant correlation existed between P120 expression and either tumour size or hormone receptors. A significant correlation was found between P120 expression and histological grade, degree of aneuploidy, S-phase fraction, degree of nodal involvement, and stage of disease. P120 is a biological marker indicative of tumour aggressiveness and may play an important role in determining which patients would most benefit from adjuvant therapy.