p21(Waf1/Cip1/Sdi1) mediates retinoblastoma protein degradation.

Damage-induced G1 checkpoint in mammalian cells involves upregulation of p53, which activates transcription of p21(Waf1) (CDKN1A). Inhibition of cyclin-dependent kinase (CDK)2 and CDK4/6 by p21 leads to dephosphorylation and activation of Rb. We now show that ectopic p21 expression in human HT1080 fibrosarcoma cells causes not only dephosphorylation but also depletion of Rb; this effect was p53-independent and susceptible to a proteasome inhibitor. CDK inhibitor p27 (CDKN1B) also caused Rb dephosphorylation and depletion, but another CDK inhibitor p16 (CDKN2A) induced only dephosphorylation but not depletion of Rb. Rb depletion was observed in both HT1080 and HCT116 colon carcinoma cells, where p21 was induced by DNA-damaging agents. Rb depletion after DNA damage did not occur in the absence of p21, and it was reduced when p21 induction was inhibited by p21-targeting short hairpin RNA or by a transdominant inhibitor of p53. These results indicate that p21 both activates Rb through dephosphorylation and inactivates it through degradation, suggesting negative feedback regulation of damage-induced cell-cycle checkpoint arrest.

Hydrographics and the timing of infectious salmon anemia outbreaks among Atlantic salmon (Salmo salar L.) farms in the Quoddy region of Maine, USA and New Brunswick, Canada.

Infectious salmon anemia (ISA) has caused severe morbidity and mortality in farmed Atlantic salmon in North America, Norway, Scotland and the Faroe Islands. The Quoddy region of Maine, United States of America (USA), and New Brunswick (NB), Canada is characterized by extensive tidal mixing and close proximity between farms. This region is also prone to recurrent appearances of ISA, though control measures limit disease spread and severity on infected farms. We conducted a retrospective longitudinal analysis of the apparent impact of hydrographics on the incidence and timing of ISA outbreaks on Atlantic salmon (Salmo salar L.) farms in the Quoddy region from May 2002 to August 2004. A time-series cross-sectional regression of 32 farms over 28 months demonstrated a limited, but statistically significant, spatio-temporal clustering of ISA outbreaks linked hydrographically. New outbreaks correlated temporally with those occurring on-site 1 and 3 months prior, and those occurring within one tidal-excursion upstream the same month. Other risk factors included holdover of previous year-class fish, wharf sharing, and possibly harvests of cages infected in previous months. Conclusions suggest that tidal dispersion does play a role in ISAV transmission in the Quoddy region. Dispersal of free virus and/or tidal distribution of lice or other hydrographically influenced vectors or fomites could all contribute to the spatio-temporal patterns described.

Polyamine depletion in human melanoma cells leads to G1 arrest associated with induction of p21WAF1/CIP1/SDI1, changes in the expression of p21-regulated genes, and a senescence-like phenotype.

The cell cycle regulatory events that interface with polyamine requirements for cell growth have not yet been clearly identified. Here we use specific inhibitors of polyamine biosynthetic enzymes to investigate the effect of polyamine pool depletion on cell cycle regulation. Treatment of MALME-3M cells with either the ornithine decarboxylase inhibitor alpha-difluoromethylornithine or the S-adenosylmethionine decarboxylase inhibitor MDL-73811 lowered specific polyamine pools and slowed cell growth but did not induce cell cycle arrest. By contrast, treatment with the combination of inhibitors halted cell growth and caused a distinct G1 arrest. The latter was associated with marked reduction of all three polyamine pools, a strong increase in p21(WAF1/CIP1/SDI1) (p21), and hypophosphorylation of retinoblastoma protein. All effects were fully prevented by exogenous polyamines. p21 induction preceded p53 stabilization in MALME-3M cells and also occurred in a polyamine-depleted, p53-nonfunctional melanoma cell line, indicating that p21 is induced at least in part through p53-independent mechanisms. Conditional overexpression of p21 in a fibrosarcoma cell line was shown previously to inhibit the expression of multiple proliferation-associated genes and to induce the expression of genes associated with various aspects of cell senescence and organism aging. Polyamine depletion in MALME-3M cells was associated with inhibition of seven of seven tested p21-inhibited genes and with induction of 13 of 14 tested p21-induced genes. p21 expression is also known to induce a senescence-like phenotype, and phenotypic features of senescence were observed in polyamine-depleted MALME-3M cells. Cells increased in size, appeared more granular, and expressed senescence-associated beta-galactosidase. Cells released from the polyamine inhibition lost the ability to form colonies, failed to replicate their DNA, and approximately 25% became bi- or multinucleated. These events parallel the outcome of prolonged p21 induction in fibrosarcoma cells. The results of this study indicate that polyamine pool depletion achieved by specific biosynthetic enzyme inhibitors causes p21-mediated G1 cell cycle arrest followed by p21-mediated changes in gene expression, development of a senescence-like phenotype, and loss of cellular proliferative capacity.

A senescence-like phenotype distinguishes tumor cells that undergo terminal proliferation arrest after exposure to anticancer agents.

Exposure of human tumor cell lines to different chemotherapeutic drugs, ionizing radiation, and differentiating agents induced morphological, enzymatic, and ploidy changes resembling replicative senescence of normal cells. Moderate doses of doxorubicin induced this senescence-like phenotype (SLP) in 11 of 14 tested cell lines derived from different types of human solid tumors, including all of the lines with wild-type p53 and half of p53-mutated cell lines. SLP induction seemed to be independent from mitotic cell death, the other major effect of drug treatment. Among cells that survived drug exposure, SLP markers distinguished those cells that became terminally growth-arrested within a small number of cell divisions from the cells that recovered and resumed proliferation. SLP induction in breast carcinoma cells treated with retinoids in vitro or in vivo was found to correlate with permanent growth inhibition under the conditions of minimal cytotoxicity, suggesting that this response may be particularly important for the antiproliferative effect of differentiating agents. The senescence-like program of terminal proliferation arrest may provide an important determinant of treatment outcome and a target for augmentation in cancer therapy.

Role of p53 and p21waf1/cip1 in senescence-like terminal proliferation arrest induced in human tumor cells by chemotherapeutic drugs.

Exposure of human tumor cell lines to moderate doses of anticancer agents induces terminal proliferation arrest accompanied by morphologic and enzymatic changes that resemble senescence of normal cells. We have investigated the role of p53 and p21waf1/cip1 in the induction of this response in drug-treated tumor cells. Doxorubicin treatment induced the senescence-like phenotype (SLP) and its associated terminal growth arrest in wild-type HCT116 colon carcinoma cells; this response was strongly decreased but not abolished in HCT116 lines with homozygous knockout of p53 or p21. Transduction of HT1080 fibrosarcoma cells with a genetic inhibitor of p53 also decreased the induction of SLP and increased drug-induced mitotic cell death. To determine if drug-stimulated p21 expression was responsible for senescence-like growth arrest, we have expressed different levels of p21 from an inducible promoter. While high-level overexpression of p21 was sufficient to induce SLP in HT1080 cells, the levels of p21 expressed in doxorubicin-treated cells could account for only a fraction of doxorubicin-induced SLP. Our results indicate that p53 and p21 act as positive regulators of senescence-like terminal proliferation arrest, but their function is neither sufficient nor absolutely required for this treatment response in tumor cells.

p21Waf1/Cip1/Sdi1-induced growth arrest is associated with depletion of mitosis-control proteins and leads to abnormal mitosis and endoreduplication in recovering cells.

Induction of a cyclin-dependent kinase inhibitor p21Waf1/ Cip1/Sdi1 is an integral part of cell growth arrest associated with senescence and damage response. p21 overexpression from an inducible promoter resulted in senescence-like growth arrest in a human fibrosarcoma cell line. After release from p21-induced growth arrest, cells re-entered the cell cycle but displayed growth retardation, cell death and decreased clonogenicity. The failure to form colonies was associated with abnormal mitosis and endoreduplication in the recovering cells and was correlated with the induced level of p21 and the duration of p21 induction. p21 induction was found to inhibit the expression of multiple proteins involved in the execution and control of mitosis. p21-induced depletion of the cellular pools of mitosis-control proteins was followed by asynchronous resynthesis of such proteins after release from p21, which explains the observed mitotic abnormalities. Genetic destabilization in cells recovering from p21-induced growth arrest may conceivably play a role in carcinogenesis and tumor progression.

Inducible retroviral vectors regulated by lac repressor in mammalian cells.

We have developed lac repressor-regulated retroviral expression vectors that are induced by beta-galactosides upon stable transduction in mammalian cells. These vectors, derived from a Moloney-virus-based vector LNCX, contain an internal Rous sarcoma virus (RSV) or cytomegalovirus (CMV) promoter, coupled with 2 to 4 lac operator sequences and placed in anti orientation relative to the retroviral long terminal repeat (LTR). Three different vectors were tested in stably infected mass populations of mouse and human cells expressing the lac repressor, in parallel with the constitutively expressed LNCX vector. The highest expression levels from these vectors ranged from 1-4% to 25-33% of the LNCX level, and the induction by beta-galactosides ranged from 6-11-fold to 29-54-fold. These vectors should be suitable for studies requiring efficient gene transfer and regulated expression in mass populations of stably transduced mammalian cells.

Estrous cycle modulation of O6-alkylguanine-DNA alkyltransferase expression in rat mammary epithelial cells.

N-methyl-N-nitrosourea (MNU)-induced rat mammary tumor incidence and tumor number per rat, is directly correlated with an increase in the circulating level of estrogen(s) at the time of carcinogen administration and subsequent mammary epithelial O6-methylguanine content. We report that, expression of O6-alkyltransferase (AGT) is also regulated by reproductive hormones in a tissue specific manner. The level of mammary epithelial cell AGT activity on estrus (0.47 pmol/mg protein) and proestrus (0.32) was significantly higher than on metestrus (0.14) (P < 0.05, estrus vs. metestrus). However, no change was observed in liver AGT activity (0.52 pmol/mg protein). In contrast, the mean level of AGT protein was not significantly different between tumors from rats injected with MNU on different days of the estrous cycle. In conclusion, the different tumor biologies resulting from carcinogen injection on different days of the estrous cycle may be partially explained by variation in levels of DNA repair activity. However, the cells in the resulting tumors did not continue an obligatory differential expression of the AGT activity consistent with their stage of initiation.

Early diagnosis of scoliosis based on school-screening.

A total of 8165 schoolchildren were screened for scoliosis by two teams of orthopaedic surgeons using the same procedures and criteria. Of 790 children who had positive physical signs, 689 were examined roentgenographically. Using 5 and 10 degrees as cut-off points, the prevalence of scoliosis was 6.58 and 2.4 per cent, respectively. A follow-up study of children who had been reported to have scoliosis at the age of eleven months showed that only half of them actually had scoliosis.

Alteration of skin protein kinase C alpha protein and mRNA levels during induced mouse hair growth.

Protein kinase C (PKC) has been implicated in regulation of hair growth. In this study, the role of PKC alpha in induced mouse hair growth was studied. Hair growth in C57BL6 mice, a well known model for hair growth research, was induced by plucking the telogen hair. PKC alpha protein levels during the induced hair growth cycle were analyzed by Western immunoblot and mRNA levels were measured by RT-PCR. At 1 day and 4 days postdepilation, when the induced hair cycle was in early and midanagen, the PKC alpha protein level was decreased. At 10 days after depilation, when the induced hair cycle was in mature anagen, the PKC alpha protein level was increased. At 17 days after plucking the hair, when the induced hair cycle was in early catagen, PKC alpha protein returned to the control level. PKC alpha mRNA was relatively unchanged at 1 day and 4 days after plucking the hair but significantly elevated at 10 days postdepilation. At 17 days after hair growth induction, PKC alpha mRNA reverted to the control level. These results suggest that: 1) in early and mid anagen of the induced hair growth cycle, PKC alpha was downregulated posttranscriptionally. This downregulation may play a role in the induction of hair growth; 2) in mature anagen of induced hair growth cycle, PKC alpha was overexpressed, and this overexpression may play a part in maintaining the hair growth. Since the expression of PKC alpha was roughly correlated with mouse skin pigmentation, we hypothesize that PKC alpha may regulate hair growth partially through modulation of skin melanogenesis.

Optimization for solidification of low-level-radioactive resin using Taguchi analysis.

Solidification of low-level-radioactive (LLW) resin was optimized using Taguchi analytical methodology. The ingredients in LLW mortar which caused the solidification of cement were evaluated through consecutive measurements of the effects of various concentrations of ingredients. Samples selected according to Taguchi's method were separated into 18 different categories and measured at the 7th, 21st, and 28th day after fabrication on developing effects. Evaluations of the various samples focused on whether the compressive and bending strength fulfilled the special criteria of the Taiwan Power Company (TPC). Similar results indicated that both furnace slag and fly ash were the dominant material resulting from the solidification of LLW mortar. The superior combination was obtained as furnace slag 24 wt.%, fly ash 24 wt.%, and cement 8 wt.% to mix 24 wt.% of resin with 20 wt.% of water, to fulfill the contemporary requirements of TPC.

The phoBR operon in Escherichia coli K-12.

The phoB and phoR genes encode a transcription activator and a sensory protein of the phosphate regulon, respectively. It is shown here that they were transcribed as an operon in which the phoB gene was promoter proximal. Although an operon structure was suggested previously (K. Makino, H. Shinagawa, M. Amemura, and A. Nakata, J. Mol. Biol. 190:37-44 and 192:549-556, 1986), previous results showed only that phoR gene expression during phosphate limitation is dependent on the upstream phoB promoter. The phoR gene could still have had its own promoter for expression in the presence of phosphate. Two polar transposon-induced mutations are described which simultaneously abolished phoB and phoR gene function in cis; one mutation mapped in the phoB gene, and the other mapped upstream of the phoB gene. These results demonstrate an operon structure, in which phoR gene function required expression from the phoB promoter. Unexpectedly, an antisense pho omega Mu d1(lacZ) insertion within the promoter-proximal end of the phoB gene expressed the lacZ reporter gene, thus allowing for the possibility that the phoBR operon is regulated by an antisense RNA.

If not apoptosis, then what? Treatment-induced senescence and mitotic catastrophe in tumor cells.

Inhibition of the program of apoptosis has been reported to have little or no effect on clonogenic survival after treatment with drugs or radiation in several tumor cell lines. A decrease in apoptosis is compensated in such cell lines by an increase in the fractions of cells that undergo permanent growth arrest with phenotypic features of cell senescence, or die through the process of mitotic catastrophe. Most of the tested tumor cell lines have retained the capacity of normal cells to undergo accelerated senescence after treatment with DNA-interactive drugs, ionizing radiation, or cytostatic agents. p53 and p21(Waf1/Cip1/Sdi1) act as positive regulators of treatment-induced senescence, but they are not required for this response in tumor cells. The senescent phenotype distinguishes tumor cells that survived drug exposure but lost the ability to form colonies from those that recover and proliferate after treatment. Although senescent cells do not proliferate, they are metabolically active and may produce secreted proteins with potential tumor-promoting activities. The expression of such proteins is mediated at least in part by the induction of p21(Waf1/Cip1/Sdi1). The other anti-proliferative response of tumor cells is mitotic catastrophe, a form of cell death that results from abnormal mitosis and leads to the formation of interphase cells with multiple micronuclei. Mitotic catastrophe is induced by different classes of cytotoxic agents, but the pathways of abnormal mitosis differ depending on the nature of the inducer and the status of cell-cycle checkpoints. Mitotic catastrophe can also develop as a consequence of aberrant reentry of tumor cells into cell cycle after prolonged growth arrest. Elucidation of the factors that regulate different aspects of treatment-induced senescence and mitotic catastrophe should assist in improving the efficacy and decreasing side effects of cancer therapy.

Applications of green fluorescent protein as a marker of retroviral vectors.

The Green Fluorescent Protein (GFP) of Aequorea victoria is used as a vital fluorescent tag for the detection and isolation of genetically modified cells. Several modified variants of GFP were tested as marker genes in retroviral vectors containing different backbones and promoter combinations. Constructs allowing for reliable detection of GFP fluorescence and the expression of a cotransduced gene from a strong promoter were identified. Cells harboring such constructs are detectable by flow cytometry, fluorescence microscopy and multi-well fluorescence reading. GFP expression in transduced cells is stable both in vitro and in vivo, and long-term dynamics of GFP-positive fractions in a mixed population can be used to monitor the biological effects of a cotransduced gene. Selection of cells with the highest GFP fluorescence enriches for multiply infected cells. The use of different GFP variants allows one to monitor simultaneously two cell populations transduced with vectors carrying GFPs that differ in their fluorescence intensity or spectral properties and to identify doubly transduced cells. In addition, transcription of an inducible promoter positioned in the opposite orientation to GFP can be monitored by the inhibition of GFP fluorescence. Thus, GFP provides a useful marker for gene transfer by retroviral vectors and extends the range of applications for retroviral transduction.

Effects of p21Waf1/Cip1/Sdi1 on cellular gene expression: implications for carcinogenesis, senescence, and age-related diseases.

Induction of cyclin-dependent kinase inhibitor p21(Waf1/Cip1/Sdi1) triggers cell growth arrest associated with senescence and damage response. Overexpression of p21 from an inducible promoter in a human cell line induces growth arrest and phenotypic features of senescence. cDNA array hybridization showed that p21 expression selectively inhibits a set of genes involved in mitosis, DNA replication, segregation, and repair. The kinetics of inhibition of these genes on p21 induction parallels the onset of growth arrest, and their reexpression on release from p21 precedes the reentry of cells into cell cycle, indicating that inhibition of cell-cycle progression genes is a mechanism of p21-induced growth arrest. p21 also up-regulates multiple genes that have been associated with senescence or implicated in age-related diseases, including atherosclerosis, Alzheimer's disease, amyloidosis, and arthritis. Most of the tested p21-induced genes were not activated in cells that had been growth arrested by serum starvation, but some genes were induced in both forms of growth arrest. Several p21-induced genes encode secreted proteins with paracrine effects on cell growth and apoptosis. In agreement with the overexpression of such proteins, conditioned media from p21-induced cells were found to have antiapoptotic and mitogenic activity. These results suggest that the effects of p21 induction on gene expression in senescent cells may contribute to the pathogenesis of cancer and age-related diseases.