Cognitive-behavioral stress management intervention decreases the prevalence of depression and enhances benefit finding among women under treatment for early-stage breast cancer.

The authors tested effects of a 10-week group cognitive-behavioral stress management intervention among 100 women newly treated for Stage 0-II breast cancer. The intervention reduced prevalence of moderate depression (which remained relatively stable in the control condition) but did not affect other measures of emotional distress. The intervention also increased participants' reports that having breast cancer had made positive contributions to their lives, and it increased generalized optimism. Both remained significantly elevated at a 3-month follow-up of the intervention. Further analysis revealed that the intervention had its greatest impact on these 2 variables among women who were lowest in optimism at baseline. Discussion centers on the importance of examining positive responses to traumatic events--growth, appreciation of life, shift in priorities, and positive affect-as well as negative responses.

Analysis of viral infection and viral and cellular DNA and proteins by flow cytometry.

Viruses are obligate intracellular parasites that require the host cell replication, transcription, and translation machinery for reproduction. Each viral group provides a unique series of viral-cellular interactions. Studies have provided insight not only into viral replication and control of host functions, but also into cellular functions such as eukaryotic replication, transcription, and translation as well as the regulation of these events. This unit presents a protocol for flow cytometric monitoring of viral infection and quantitating viral-cellular events. The availability of monoclonal and/or polyclonal antibodies directed to both viral and cellular proteins offers the ability to assay a specific molecule in the intact fixed cell and the opportunity to correlate viral events with cellular processes such as progression through the cell cycle.

Simian virus 40 induces multiple S phases with the majority of viral DNA replication in the G2 and second S phase in CV-1 cells.

The infection of permissive monkey kidney cells (CV-1) with simian virus 40 induces G1 growth-arrested cells into the cell cycle. After completion of the first S phase and movement into G2, mitosis was blocked and the cells entered another DNA synthesis cycle (second S phase). Growth-arrested CV-1 cells replicated significant amounts of viral DNA in the G2 phase with the majority of synthesis occurring during the second S phase. When mimosine-blocked (G1/S) infected cells were released into the cell cycle, a major portion of the viral DNA was detected in G2 with the largest accumulation in the second S phase. The total DNA produced per infected cell was 10-12C with approximately 0.5-2C of viral DNA replicated per cell. Therefore the majority of the DNA per cell was cellular, 4C from the first S phase and approximately 4-6C from the second cellular synthesis phase.

A subset of cells expressing SV40 large T antigen contain elevated p53 levels and have an altered cell cycle phenotype.

Cells transformed by the simian virus 40 (SV40) large T antigen (Tag) contain elevated levels of cellular p53 protein. To quantify this relationship, levels of p53 were measured in NIH 3T3 cells that expressed different concentrations of Tag. Using immunoblotting, average p53 levels were shown to increase linearly with Tag concentrations in these cell lines. Single-cell measurements were also performed using flow cytometry to measure p53 immunofluorescence. Surprisingly, the flow cytometry experiments showed that two distinct cell populations, based on p53 content, were present in cells expressing high levels of Tag. One cell population contained elevated p53 levels. A second population did not contain elevated p53, even though high concentrations of Tag were present in the cells. This latter cell population did not appear to arise because of mutations in either Tag or p53. The two cell populations also had phenotypic differences. In exponentially growing cells, Tag alters the cell cycle distribution (decreases the percentage of G1 phase cells and increases the percentages of S and G2 + M phase cells). This phenotype was maximum in the cell population containing elevated p53. A lesser phenotype was found in the cell population that did not contain elevated p53. These data show, firstly, that cells can express significant levels of Tag and not contain elevated levels of p53 and, secondly, that elevated p53 correlates with the altered cell cycle distribution produced by Tag in growing cells.

SV40 small T antigen enhances progression to >G2 during lytic infection.

The infection of monkey kidney (CV-1) cells with simian virus 40 (SV40) stimulates the cells into successive rounds of DNA synthesis without an intervening mitosis, leading to the acquisition of a >G2 DNA content. To elucidate the role of small t antigen in cell cycle progression and in viral replication during infection, studies were performed using an SV40 mutant (dl888) that lacks the ability to produce small t. Initially dl888-infected cells move through the first S phase at roughly the same rate as wild-type infected cells. Upon reaching G2, however, the dl888-infected cells progressed to >G2 at a reduced rate relative to wild-type. The slower rate of entry into >G2 of dl888-infected cells is associated with a decrease in total pRb and an increase in the ratio of hypophosphorylated to hyperphosphorylated pRb. The expression of cyclin D1 and p27(kip1) were elevated in dl888-infected cells compared to wild-type-infected CV-1 cells. Taken together, these results indicate that small t antigen plays a role in stimulating entry into >G2 in SV40-infected CV-1 cells, possibly by affecting the regulation of key cell cycle proteins.

Comparison of bone marrow extracellular matrices.

We have compared the structure and composition of adult and fetal bovine bone marrow extracellular matrices. In contrast to fetal bone marrow, adult bone marrow has more oval fenestration and accumulation of adipocytes as well as lower protein content. These differences could be due to remodeling of bone marrow tissue as it develops. Zymogram analysis of matrix metalloproteinase (MMP) and tissue inhibitor of MMP (TIMP) activities showed that fetal, but not adult bone marrow extract contained a 96-kDa MMP and TIMP-1 and -2. These activities may contribute to the structural differences between adult and fetal bone marrow tissues.

Concerns about breast cancer and relations to psychosocial well-being in a multiethnic sample of early-stage patients.

Much work on psychosocial sequelae of breast cancer has been guided by the assumption that body image and partner reaction issues are focal. In a tri-ethnic sample of 223 women treated for early-stage breast cancer within the prior year, the authors assessed a wider range of concerns and relations to well-being. Strongest concerns were recurrence, pain, death, harm from adjuvant treatment, and bills. Body-image concerns were moderate; concern about rejection was minimal. Younger women had stronger sexual and partner-related concerns than older women. Hispanic women had many stronger concerns and more disruption than other women. Life and pain concerns and sexuality concerns contributed uniquely to predicting emotional and psychosexual disruption; life and pain concerns and rejection concerns contributed to predicting social disruption. In sum, adaptation to breast cancer is a process bearing on several aspects of the patient's life space.

Identification of SV40 T-antigen mutants that alter T-antigen-induced chromosome damage in human fibroblasts.

The SV40 T antigen causes numerical (aneuploidy) and structural (aberrations) chromosome damage when expressed in human diploid fibroblasts. This chromosome damage precedes the acquisition of neoplastic traits such as anchorage independence, colony formation in reduced serum growth factors, immortalization, or tumorigenicity. Therefore, chromosome damage may be important in acquiring these traits because it could provide a mutational mechanism. To determine how the T antigen causes chromosome damage, point mutations were constructed that altered previously defined biochemical functions of the T protein. Mutant T antigen constructs were introduced into human diploid fibroblasts and selected by using G418. Clones of G418r cells that expressed mutant T antigens were expanded and scored for chromosome damage. Most of these mutant T antigens caused [corrected] levels of chromosome damage similar to those caused by [corrected] the wild-type T antigen. However, some T-antigen mutants induced fewer chromosome changes. A subset of these clones that induced less chromosome damage than wild-type T were examined further. Mutant T-antigen protein levels from this subset were quantified with flow cytometry and compared with wild-type protein expression levels. Mutations of T antigen shown previously to form less stable complexes with p53 caused less chromosome damage. A mutation in the zinc finger domain of T antigen also caused less chromosome damage. Interestingly, a mutant that caused loss of the ATPase activity of T antigen caused an increase in endoreduplicated cells. Also, a correlation was noted between cells expressing very low levels of T antigen (below detection limits when using flow cytometry) and an undamaged karyotype. This correlation indicates that there is a threshold level of T-antigen expression that induces chromosome damage and that expression levels on a per-cell basis rather than on a population basis should be considered in subsequent studies.

Activities of SV40 T antigen necessary for the induction of tetraploid DNA content in permissive CV-1 cells.

To determine the role of SV40 T antigen in stimulating multiple rounds of DNA synthesis in permissive cells, CV-1 cells were transfected with plasmids expressing mutant or wt T antigen in the presence or absence of the SV40 origin of replication. Induction of cells with > G2 DNA content (tetraploid DNA content) and levels of T antigen protein were detected and analyzed by flow cytometry. The mutant T antigen proteins demonstrated the expected phenotypes as determined by immunoprecipitation. Elevated levels of T antigen protein were detected in each transfection, but full-length T antigen alone was responsible for the tetraploid DNA content. The studies show that full-length T antigen with point mutations to reduce binding to the cellular proteins p53 and/or Rb were capable of inducing > G2 DNA content though the induction by these mutants was greatly enhanced by the presence of the SV40 origin of replication. Truncated T antigen (aa 1-259) could induce cells with tetraploid DNA content only in the presence of the SV40 origin of replication and the absence of Rb binding. These studies suggest that multiple functions of T antigen are involved in the stimulation of the second round of cellular DNA synthesis.

Okadaic acid induces appearance of the mitotic epitope MPM-2 in SV40-infected CV-1 cells with a >G2-phase DNA content.

Simian virus 40 (SV40) infection of quiescent monkey kidney cells stimulates two successive rounds of cellular DNA synthesis without an intervening mitosis. This uncoupling of S phase and mitosis indicates that SV40 modulates pathways regulating the G2-to-M phase transition. To examine the integrity of mitotic initiation pathways in infected cells that have bypassed mitosis, SV40-infected CV-1 cells were treated with okadaic acid (OA), a known inducer of premature mitosis in other cell types. OA treatment triggered the appearance of the mitotic marker MPM-2 in SV40-infected CV-1 cells progressing through either the first (diploid) or second (tetraploid) S phases. These results demonstrate that a subset of mitotic pathways are intact but inactive in SV40-infected cells that have bypassed mitosis and initiated tetraploid S phase.

Induction of tetraploid DNA content by mutant simian virus 40 T antigen that has reduced complex formation with p53.

The 402 mutants (DE, DH, DN) of simian virus (SV) 40 form reduced levels of p53-T antigen complexes or no complexes in lytically infected cells (CV-1 cells) relative to wild-type (wt) virus when assayed by immunoprecipitation. When CV-1 cells were infected with the 402 mutants, the cells were induced into multiple rounds of DNA synthesis without mitosis, resulting in a large population of cells with > G2 (tetraploid) DNA content similar to wt virus. The levels of T antigen and p53 per cell that were determined by flow cytometry were similar to wt lytically infected cells, with the levels of T antigen increasing as the infection proceeded. The p53 increased as the levels of T antigen increased, similar to a wt infection. These studies demonstrate that, in lytically infected cells with reduced p53-T antigen complex formation, the cells are induced into multiple rounds of DNA synthesis.

Increase in total protein following infection of CV-1 cells with SV40 virus as assayed by flow cytometry.

The changes in cell size and total protein were determined for G1-arrested, contact-inhibited CV-1 cells infected with Simian virus 40 (SV40). The assays used were the Biorad total protein assays (Bradford and DC protein assays) on a standard number of cells, total protein as assayed by fluorescein isothiocyanate (FITC) and SR101 by flow cytometry, orthoganol (90 degrees) light scatter by flow cytometry, and direct microscopic measurement with an ocular micrometer. Uninfected CV-1 cells and two cell lines with variations in DNA content (diploid vs. tetraploid) were used as controls for the studies presented. The results demonstrated a 40-60% increase in total protein at 32 to 42 h postinfection. These increases were similar to values obtained due to cellular changes resulting from viral replication and cell death.

Increased p53 protein associated with aging in human diploid fibroblasts.

Molecular changes associated with cellular aging in a strain of human diploid fibroblasts, IMR-90, were addressed by analyzing the expression of the tumor suppressor protein, p53. In all studies, IMR-90 cultures were characterized as "young" or "near-senescent" based on morphology, rate of population doubling, capacity for DNA synthesis, and presence of established markers for senescence. When p53 was immunoprecipitated by monoclonal antibodies and detected by Western immunoblot analysis, more protein per cell was detected in the near-senescent cultures. A greater than 10-fold increase in p53 protein was measured with the PAb 1801 (N-terminal-specific) anti-p53 antibody, whereas PAb 122 (C-terminal-specific) measured a 5-fold increase. Although near-senescent cultures demonstrated a higher level of p53 than young cells, these cultures had similar charges and molecular weight p53 isoforms when analyzed by two-dimensional Western blots. When p53 RNA was compared to total RNA there was a decrease in p53 RNA with age, but on a per cell basis p53 RNA was elevated. These results provide evidence for transcriptional regulation of p53 during aging and support the hypothesis that elevated levels of p53 protein may play a role in cellular senescence.

Induction of tetraploid DNA content by simian virus 40 is dependent on T-antigen function in the G2 phase of the cell cycle.

Previous experiments with the simian virus 40 mutant tsA357R-K (tsA30) demonstrated a T-antigen function that is required for production of cells with a greater-than-G2-phase DNA content. In this study, temperature shift experiments indicated that the temperature-sensitive function of tsA357R-K, which is necessary for entry into the greater-than-G2 phase, is not required in G1 or S but must be supplied in the G2 phase.

DNA content distribution of mouse cells following infection with polyoma virus.

Infection of primary to tertiary mouse embryo fibroblasts or mouse kidney cells with polyoma virus leads to stimulation of cellular DNA synthesis. When either confluent or growing mouse cells were infected, the monolayer cells were found to accumulate cells with a DNA content of S and G2/M phases of the cell cycle as assayed by flow cytometry. A similar pattern of DNA content was also observed in cells in the supernatant, which are probably cells replicating virus and dying. When compared with control cells, the infected monolayer and supernatant cells exhibited a population (5-27%) with a > G2 DNA content. The increase in DNA content of these > G2 cells was calculated to be an average of 26.7%, which is probably due to viral DNA. Polyoma contrasts with another papovavirus, SV40, which stimulates cells into DNA synthesis, with the majority of cells attaining a > G2/tetraploid DNA content, suggesting that there are differences in polyploidization between these two viruses.

Simian virus 40 prevents activation of M-phase-promoting factor during lytic infection.

Simian virus 40 (SV40) infection stimulates confluent cultures of monkey kidney cells into successive rounds of cellular DNA synthesis without intervening mitosis. As an initial step in defining the mechanisms responsible for viral inhibition of mitosis, M-phase-promoting factor (MPF) was examined in SV40-infected CV-1 cells passing from G2 phase into a second S phase. MPF is a serine-threonine protein kinase that is essential for mitosis in eukaryotic cells. In SV40-infected cells exiting G2 phase, there was a reduced amount of MPF-associated H1 kinase activity relative to that of uninfected cells passing through mitosis. Both subunits of MPF, cyclin B and the p34cdc2 catalytic subunit, were present and in a complex in infected cells. In uninfected cultures, passage through mitosis was associated with the dephosphorylation of the p34cdc2 subunit, which is characteristic of MPF activation. In contrast, the p34cdc2 subunit remained in the tyrosine-phosphorylated, inactive form in SV40-infected cells passing from G2 phase into a second S phase. These results suggest that although the MPF complex is assembled and modified normally, SV40 interferes with pathways leading to MPF activation.

A new perspective on threatened autonomy in elderly persons: the disempowering process.

This study explored factors other than medical condition and treatments which contributed to the discharge experiences of 12 rural and 9 urban patients. Interpretive research methodology included document review, observation and in-depth interviews of all key participants. The purposefully selected sample consisted of a total of 21 patients, 22 informal caregivers, and 117 professionals involved in the hospital and/or home setting. Findings document a new perspective on how patients and professionals together contribute to the patient's threatened autonomy. Lack of clarity about goals, aspirations, and purpose in life and a generally negative frame of mind in the elderly combine with professional practice approaches to create a disempowering process. Faced with the biomedical orientation and paternalism of professionals, patients with a positive mindset and sense of direction and purpose in life did not experience threat to their autonomy. The researchers conclude that empowerment strategies must encompass a patient-centred approach, which includes an understanding of the patient's mindset, goals, aspirations, and sense of purpose within a larger life context. This consideration is essential to enable elderly patients to maintain autonomy despite continued health care requirements.

Hypophosphorylated retinoblastoma gene product accumulates in SV40-infected CV-1 cells acquiring a tetraploid DNA content.

Simian virus 40 (SV40) infection of monkey kidney cells induces successive rounds of cellular DNA synthesis without intervening mitosis. To gain an understanding of the mechanisms responsible for disruption of cell cycle control during lytic infection, pRB phosphorylation and cell cycle distribution were examined following SV40 infection of CV-1 cells. The hypophosphorylated pRB present in confluent CV-1 cells was phosphorylated within 14 h following SV40 infection. Phosphorylated pRB then remained the predominant form as cells progressed from late G1 through S phase. Hypophosphorylated pRB reappeared as cells moved through G2 and acquired a tetraploid (> G2) DNA content. The reappearance of hypophosphorylated pRB in a population with decreasing numbers of cells in G1 phase and increasing numbers of cells in > G2 suggests that accumulation of hypophosphorylated pRB may be involved in T antigen-induced tetraploidy.