Background: Analyzing apoptosis has been an integral component of many biological studies. However, currently available methods for quantifying apoptosis have various limitations including multiple, sometimes cell-damaging steps, the inability to quantify live, necrotic and apoptotic cells at the same time, and non-specific detection (i.e. "false positive"). To overcome the shortcomings of current methods that quantify apoptosis in vitro and to take advantage of the 96-well plate format, we present here a modified ethidium bromide and acridine orange (EB/AO) staining assay, which may be performed entirely in a 96-well plate. Our method combines the advantages of the 96-well format and the conventional EB/AO method for apoptotic quantification.
The cholesterol-lowering medications, statins, inhibit cellular proliferation and induce apoptosis in an array of cancer cell lines, including melanoma. We investigated the apoptotic mechanism of lovastatin on human melanoma cell lines in vitro. The cytotoxicity of statins on multiple cell lines was examined by Cell Titer 96 Aqueous One solution cell proliferation assay (MTS assay). Apoptosis was assayed by ethidium bromide and acridine orange morphologic assays, an Annexin V apoptosis detection kit and active caspase 3 assays. Farnesyl pyrophosphate and geranylgeranyl pyrophosphate add-back experiments were performed to better define the molecular mechanisms mediating lovastatin cytotoxicity. Lovastatin caused cytotoxicity in human and murine melanoma cells, but did not induce toxicity in an epidermoid carcinoma cell line A431. For human melanoma cells, lovastatin precipitated cell rounding, increased the percentage of apoptotic cells detected by ethidium bromide and acridine orange staining and by the Annexin V apoptosis detection kit, and resulted in a 50-fold increase in active caspase 3, corroborating that lovastatin induced apoptosis. Adding back geranylgeranyl pyrophosphate, but not farnesyl pyrophosphate, reversed the effects of lovastatin in A375 cells. Of the five statins tested, pravastatin was least effective in killing melanoma cells. Lovastatin induced caspase-dependent apoptosis in multiple melanoma cell lines via a geranylation-specific mechanism. This study supports a possible role of lovastatin as a therapeutic, adjuvant or chemopreventive agent for melanoma.
Hyperthermia, the procedure of exposing cells to a temperature between 42 degrees and 49 degrees C, has been shown to be a promising approach for cancer treatment. To understand the underlying mechanisms of hyperthermic killing of cancer cells, it is critical to have an accurate temperature measurement technique and a heating method with high reproducibility. To this end, we have developed a method using fine thermocouples with fast response time to measure the temperatures in multiple wells of a 96-well plate. The accuracy of temperature measurement was +/- 0.2 degree C. Such a capability allows a complete record of the time and temperature of the treatment procedure and helps define an accurate thermal dose. We have also compared several methods for heating 96-well plates and found that use of copper blocks in contact with the lower surface of the 96-well plate in an incubator provides a highly reproducible heating method. The common method of using water bath to heat cells in vitro resulted in a decrease of cell viability even at the control temperature of 37 degrees C and a decrease in the reproducibility of certain biological assays. In summary, using these improved techniques, proposed thermal dose can be defined more precisely, and highly reproducible heating in vitro can be achieved.
PurposeTreating disease with hyperthermia dates back to 3000 BC when Egyptian physicians explored the therapeutic benefits of heating diseased tissues. There has been renewed interest in using hyperthermia to treat cancer over the last 30 years. Hyperthermia as an adjuvant anti-cancer therapy has demonstrated improved clinical outcomes and increased response rates when combined with conventional treatments of tumors. This project investigated the molecular mechanisms of hyperthermia induced cell death in human melanoma cells in-vitro. Data from these experiments may help in the generation of new treatment modalities for skin cancer.MethodsCopper blocks inside incubators delivered a uniform and reproducible hyperthermia treatment to human melanoma cells grown in 96 well plates. A sophisticated measurement device with fast-response thermocouples in a 96 well format measured the thermal dose delivered to the cells. The human melanoma cells received hyperthermia treatments ranging from 15-120 mins at 41, 45 and 48°C. The treated cells were analyzed for overall viability, the mechanism of cell death, and whether there was a relationship between the thermal-doses delivered and the cell death pathway induced. Cell viability was measured in each well post treatment using an MTS based assay (CellTiter 96 Aqueous One Promega). The activity of apoptosis specific caspases 3 and 7 were measured using a homogeneous luminescent assay (Caspase-Glo3/7 Promega). Nuclear condensation and fragmentation was assessed in an eloquent microscopic approach using a modified ethidium bromide and acridine orange staining assay (EB/AO) in a 96 well format.ResultsWe consistently demonstrated that hyperthermia decreased cell viability in human melanoma cells in-vitro, and that the diminished cell viability was proportional the thermal dose delivered. Hyperthermia also induced activation of caspase 3 and 7, and increased the population of cells with condensed or fragmented nuclei. This data suggest that hyperthermia induced apoptosis in human melanoma cells.ConclusionsHyperthermia induces dose-dependent apoptosis in human melanoma cells. Further elucidation of these pathways will help in the design and discovery of novel methods for treating skin cancers.
Objectives: We conducted a prospective analysis of the relation between breast size measured by self-reported bra cup size and breast cancer risk among a cohort of premenopausal women. Design: Prospective cohort study Setting: The Nurses' Health Study II is an ongoing, prospective cohort of 116,671 American female registered nurses. The study was initiated in 1989 and enrolled women between 25 -42 years of age living within 14 states in the U.S. Participants: Bra cup size and breast cancer risk was assessed among 88,787 premenopausal women aged 29 to 47 in 1993. Bra cup size at age 20 was assessed by self-report in 1993. Women were excluded if at baseline they were postmenopausal, reported previous cancer, or did not report bra cup size. Censoring occurred if a women experienced breast cancer, reached menopause or died. Main Outcome Measures: New cases of invasive breast cancer were self-reported and confirmed by review of pathology reports. Hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated with a Cox proportional hazards model adjusting for potential risk factors for breast cancer. All statistical tests were twosided. Results: During 621,097 total years of follow-up, 893 women developed incident invasive breast cancer. For women with a body mass index (BMI) below 25, those with a bra cup size of "B" (covariate adjusted HR=1.22, 95% CI 1.00-1.49) and "D or larger" (covariate adjusted HR=1.77, 95% CI 1.13-2.77) had an increased risk of breast cancer relative to "A or smaller" (Ptrend = 0.01). There was no important association among women with a BMI of 25 or higher. Stratifying by BMI at age 18 using a cut point of 21 gave similar results. Conclusion: Larger bra cup size at a younger age is associated with an increased risk of premenopausal breast cancer, though this association is limited to leaner women.
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