Absence of p16 gene (CDKN2) deletions in microdissected primary breast carcinoma specimens.

BACKGROUND: The p16 gene (CDKN2), a tumor suppressor gene located on chromosome 9p21, has been demonstrated to be mutated or deleted with high frequency in a variety of tumor cell lines, including breast. While previous studies have not demonstrated CDKN2 mutations in primary breast carcinomas, it is possible that gene deletion in neoplastic DNA was marked by the presence of contaminating normal stromal DNA in breast carcinoma specimens. METHODS: We investigated the incidence of homozygous deletion of CDKN2 by analyzing 20 microdissected pure populations of primary breast carcinoma cells. Using polymerase chain reaction (PCR) techniques, the entire coding region and intervening introns of CDKN2 were amplified. The PCR products were resolved by agarose gel electrophoresis and single-strand conformation polymorphism (SSCP) analysis. RESULTS: We detected no deletions or mutations of the p16 gene. CONCLUSIONS: CDKN2 is not deleted with high frequency in primary breast carcinomas, and the p16 gene does not play a role in breast carcinogenesis via this mechanism.

Microsatellite instability in breast cancer.

BACKGROUND: Microsatellites are short repetitive nucleotide sequences that, through mutation, can undergo either expansion or contraction. This novel mutational mechanism known as microsatellite instability may play a role in carcinogenesis. We investigated the incidence of microsatellite instability in a series of primary breast carcinoma surgical specimens. METHODS: Using polymerase chain reaction techniques followed by polyacrylamide/urea gel electrophoresis, we analyzed 46 pairs of normal and primary breast tumor samples at seven different microsatellite loci, five of which were located on chromosome 17. RESULTS: Thirteen of our 46 tumors (28.2%) demonstrated microsatellite instability. Five tumors (10.8%) were unstable at two or more loci, and of those, four (8.7%) were unstable at different loci on different chromosomes. An additional five tumors demonstrated loss of heterozygosity alone when compared with their normal counterparts. CONCLUSIONS: These findings indicate that microsatellite instability is present in primary breast cancer populations and, although the mechanism of action has yet to be elucidated, may play a role in breast carcinogenesis.

The expression of cytokine activity by fracture callus.

Cytokines, a group of proteins known to regulate hemopoietic and immune functions, are also involved in inflammation, angiogenesis, and bone and cartilage metabolism. Since all of these processes occur following bone injury, or are known to contribute to wound repair mechanisms, this investigation sought to test the hypothesis that cytokines are involved in fracture healing. Two sets of 60 male Sprague-Dawley rats underwent the production of standard closed femoral fractures. The animals were then euthanized in groups of 15 on days 3, 7, 14, and 21 postfracture. A separate control group was also used for the harvesting of intact unfractured bone. At the time of euthanasia, calluses or bone specimens were explanted to organ culture and treated with either media alone or media containing the inducing agents lipopolysaccharide or concanavalin A. A titration of conditioned medium from these cultures was then added to factor-dependent clonal cell lines that are known to be specifically responsive to interleukin-1, interleukin-6, granulocyte-macrophage colony stimulating factor or macrophage-colony stimulating factor. To confirm the identities of each of these cytokines, neutralizing antibody studies were performed. The results showed that interleukin-1 is expressed at very low constitutive levels throughout the period of fracture healing but can be induced to high activities in the early inflammatory phase (day 3). Granulocyte-macrophage colony stimulating factor showed no constitutive activity but could also be induced to high activities with lipopolysaccharide. The ability of these two cytokines to be induced declined progressively as fracture healing proceeded. Interleukin-6 showed high constitutive activity early in the healing process (day 3), and treatment with inducing agent did not increase the activity of this cytokine at this timepoint. Lipopolysaccharide did increase interleukin-6 activity in day 7 and 14 fracture calluses. Although macrophage-colony stimulating factor is thought to be involved in a variety of metabolic bone conditions, it could not be detected or induced from any of the callus samples. Moreover, none of the samples of unfractured bone showed constitutive or inducible activities for any of these cytokines. A separate experiment in which calluses and samples of unfractured bone from similar cultures were examined histologically and tested for DNA or protein synthesis at two timepoints in the culture period (days 1 and 4) showed that tissue viability was maintained. Thus the inability to detect macrophage colony-stimulating factor in fracture callus or any cytokine activity in unfractured bones was not due to cell death.(ABSTRACT TRUNCATED AT 400 WORDS)

Analysis of MTS1/CDK4 in female breast carcinomas.

Multiple factors, both environmental and genetic, are thought to play roles in breast carcinogenesis. The recently cloned multiple tumor suppressor gene (MTS1), the product of which interacts with CDK4 to regulate cell growth, has been found to be mutated with high frequency in a variety of cell lines as well as primary tumors of different histologic types. Using PCR-SSCP, we analyzed exons one (126 bp) and two (307 bp) of the MTS1 gene to determine the incidence of mutation in a population of 50 primary breast adenocarcinomas and corresponding normal tissue. Analysis of five breast tumor cell lines was also performed. We found no mutations in the MTS1 gene in the primary breast tumor samples. One cell line was found to have a homozygous deletion of the gene. Our results suggest that the MTS1 gene is not mutated with increased frequency in primary breast tumors, and thus may not play a major role in breast carcinogenesis.

Incorporation of sodium fluoride into cortical bone does not impair the mechanical properties of the appendicular skeleton in rats.

Clinical studies on the use of sodium fluoride (NaF) in osteoporotic patients have demonstrated increased spinal bone mass without a reduction in vertebral fracture incidence, and a trend towards reduced appendicular bone mass with an increase in peripheral fracture incidence. As previous reports have suggested that NaF becomes incorporated into bone's crystal structure, possibly affecting bone strength, we sought to examine the relationship among bone fluoride content, bone mass, and skeletal fragility. Twenty-one-day-old female Sprague-Dawley rats were treated with four different doses of NaF. The tibiae were subjected to histomorphometric and biochemical analyses, and the femora were tested in torsion for the properties of strength, stiffness, energy storage capacity, and angular deformation. The results showed that over 50% of the skeleton in these rats was turned over in the presence of NaF. The four different doses resulted in a linear increase in bone F concentration and suggested excellent absorption and incorporation of this drug. No changes in histomorphometric indices of bone formation or turnover were found. Despite the large fraction of bone formed during NaF treatment, and the linear increase in bone fluoride content in relation to dose, there were no changes observed in any of the mechanical properties. These results suggest that, even extensive incorporation of fluoride into bone, in the absence of an effect on bone mass or remodeling, does not significantly alter its capacity to withstand mechanical loads.