Cancer cells have shorter interphase,boost of energy at work,dandruff treatment no shampoo,how 2 treat a cold sore - Reviews

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The activation of functions that counteract the physiological shortening of telomeres in rapidly proliferating cell is prerequisite for the progression of cancer cells to full malignancy (Collado et al., 2007). Wash the suspension cells once with PBS and prepare a dilution of 0.5 x 106 cells per ml of PBS at RT.
Pre-label the slides and make a small hydrophobic barrier circle (~1.3 cm diameter) with ImmEdge pen around the area where the cells will be placed and let it dry before cytospin.
Prepare the slides mounted with the filter card and cytospin funnel in the metal holder as shown in the Figure 1.
Note: If the method will be applied to growing adherent cells on coverslip, the steps 1-4 can be skipped.
Remove the slides from the holders and let the moisture of the cells dry out for a few sec before fixation with the fixation buffer for 10 min at RT. Remove the permeabilization buffer and incubate the cells for one hour with the IF blocking buffer in moist chamber. Remove the IF blocking buffer and incubate the cells in moist chamber with the fresh IF blocking buffer containing PML antibody for 2 h at RT or overnight at 4 °C. Dehydrate the cells in coplinjar containing ethanol series, consecutively 70%, 95%, 100% EtOH, for 5 min each. Aspirate the ethanol completely and let the slides dry for a couple of minutes in the dark. Place a drop of hybridizing solution containing the PNA probe on each slide and cover it with a coverslip.
Denature the probes in hybridizing solution for 10 min at 80 °C by placing the slides on a pre-warmed hot plate.
Visualize the APBs using a confocal microscope as fluorescence signals from PML bodies co-localized with telomere signals.
Rapidly proliferating cancer cells often have shorter telomeres and higher protein expression compared to slow growing primary cells. Some principles of the described protocol have been extracted from the protocols of Dr Titia de Lange`s Lab. You are highly recommended to post your data (images or even videos) for the troubleshooting. They highlight different stages of the cell cycle, but little is known about how this affects their counts. This work was supported by grants awarded by the Swedish Cancer Society and the Karolinska Institute, Stockholm, Sweden.
For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos. This study assesses the strength of their correlations and attempts to determine the relationship between them.
The arrows show three APB foci in which the green PML signal co-localizes with the red telomere signal. To improve the method, the incubation time with the primary PML antibody should be reduced and less photobleaching telomeric probes should be used. Proliferation counts for forty-nine consecutive cases of invasive breast carcinomas were analyzed, with the same tumor area on each stain counted using digital image analysis.
APBs contain Promyelocytic Leukemia nuclear bodies (PML-NB) components such as PML, SP100 and SUMO, telomeric DNA and telomere associated proteins including the shelterin components TRF1, TRF2, POT1, TIN2, TPP1 and Rap1 (Yeager et al., 1999). In some cell lines the PML antibody gives a weak diffuse fluorescent background that is easily distinguished from the bright fluorescence of the PML bodies. Cy3-TelG usually last longer than FITC-TelC and Alexa Fluor® 488 produces less background than Alexa Fluor® 594. Once your questions are answered, you will be informed using the email address that you register with bio-protocol. The integrated optical density (IOD) of nuclei was measured as an approximation of nuclear DNA content. In cells with short telomeres and high background signals, Cy3-TelG and Alexa Fluor® 488 are recommended. The Epstein-Barr virus nuclear antigen-1 promotes telomere dysfunction via induction of oxidative stress. Telomere dysfunction and activation of alternative lengthening of telomeres in B-lymphocytes infected by Epstein-Barr virus. This is also supported by the requirement of the homologous recombination-associated MRN complex for APB formation (Wu et al., 2000). The Bloom syndrome helicase BLM interacts with TRF2 in ALT cells and promotes telomeric DNA synthesis.
Furthermore, APBs are suggested to be active sites of ATM and ATR dependent DNA repair (Nabetani et al., 2004).

With evidence from a literature review, it is suggested that the weaker correlations with MIB-1 are not explained by count imprecision or error, but relies on temporal decorrelation between cell cycle phases. Finally, the number of APBs increases in G2 phase of the cell cycle when recombination is mainly active (Grobelny et al., 2000).
Telomerase-negative immortalized human cells contain a novel type of promyelocytic leukemia (PML) body. Consequences on correlation between these proliferative markers are illustrated by mathematical models.IntroductionThe proportion of malignant cells undergoing proliferation has become an important variable in diagnostic surgical pathology, particularly in malignant neoplasms. This variable significantly influences tumor growth rate, and thus, represent tumoral aggressiveness.
More recently, it has been playing an increasingly important role in its predictive ability to guide treatment, such as in breast carcinoma1,2,3,4,5.There have been many methods of quantifying the proportion of proliferating cells. The traditional mitotic count is still used to grade a variety of tumors including breast adenocarcinomas, neuroendocrine tumors, sarcomas, meningiomas, and melanomas6,7,8,9,10,11.
It is important to remember that the proportion of proliferating cells provides incomplete information about tumor growth; the net amount of malignant cells at a certain time is the balance between the rate of newly produced cells and the rate of cell death from necrosis and apoptosis. Neglecting the negative impact of cell death, the total number of malignant cells at a given point in time is where N0 is the initial number of cells, ?t is a given interval of time, Tc is the cell cycle duration, and P is the fraction of proliferating cells. Cell death is generally assessed only as a nominal variable (present versus absent) because there is no quantitative model to measure it. In addition to the mitotic count, immunohistochemical markers of proliferation have been more recently developed and include MIB-1 which is directed at the Ki-67 antigen and targets the whole cell cycle2,12, proliferating cell nuclear antigen (PCNA) which targets the S phase13, and phosphorylated histone H3 (PhH3) which targets the mitotic phase14.While new immunohistochemical markers have been developed, mitosis remains the most important proliferative marker in clinical breast pathology. Elston and Ellis showed that if breast cancer grading protocol, which includes the mitotic score, is followed consistently, reproducible results can be obtained for breast tumor prognosis9. However mitotic score assessment is time consuming, and is not consistent due to problems of reproducibility, even amongst trained pathologists15,16,17.MIB-1 would make the visualization of proliferating cells easier16. It stains all nuclei involved in the cell cycle (G1, S, G2, and mitoses)2 and has emerged as an important independent prognostic and predictive marker in several tumors, including breast cancer1, sarcomas16,18, cutaneous melanomas11,19,20, meningiomas21, and prostate cancer22 among others.
Prevalence of the alternative lengthening of telomeres telomere maintenance mechanism in human cancer subtypes.
More specifically for breast cancer, the level of MIB-1 expression prior to neoadjuvant chemotherapy is a strong predictive factor for the potential effectiveness of the therapy, and post-therapy expression predicts disease-free survival1,3,4,5. Gallen Consensus Statements on breast cancer have supported the use of the Ki-67 index (as determined by MIB-1) in deciding whether to offer chemotherapy to patients with hormone positive, node negative cancers, and also as part of the criteria in separating luminal A from luminal B carcinomas23,24.
As a result, the Ki-67 index is increasingly being requested by medical oncologists to guide their breast cancer treatment decisions. While the function of the Ki-67 protein remains unknown, there is evidence that it has a role in cell division and ribosomal RNA synthesis25,26.
The protein name is non-descriptive, named after Kiel University in Germany where it was discovered with 67 referring to the clone number27.PhH3, in contrast to MIB-1, targets cells in the mitotic phase. Its phosphorylation at serine-10 and serine-28 are believed to be crucial for chromosome condensation and cell-cycle progression during mitosis and meiosis28,29,30.
Phosphorylation initiates in late G2 phase to early prophase and gradual dephosphorylation occurs from late anaphase to early telophase. Metaphase chromosomes are always heavily phosphorylated while interphase cells do not stain or only do so with low intensity14,29,30,31,32.
Thus, PhH3 counts should theoretically correlate with mitotic counts and has emerged as a potential immunohistochemical marker of mitotic activity.
Indeed several reports showing positive correlation between mitotic and PhH3 counts have been published20,21,33,34,35,36,37.Few studies have compared MIB-1 with PhH320,21,33,34,38,39,40.
Considerable variability in correlation between MIB-1 counts and PhH3 counts are reported, but they used methodologies that do not address the intratumoral heterogeneity observed in most solid malignant tumors which would reduce correlation between markers2,41.
The aim of this study was to measure the correlation between MIB-1 and PhH3 labeling, as well as mitotic counts, on breast adenocarcinoma. The same tumor area on each stain was assessed in order to eliminate the influence of intratumoral heterogeneity. Even if each marker assesses different intervals of the cell cycle, these markers all measure the fraction of proliferating cells and would thus be expected to strongly correlate, particularly if the durations of these intervals remain proportionally constant.
Correlations with MIB-1 and DNA content (approximated by the nuclear integrated optical density (IOD)) were assessed in order to further understand the relationships among all variables.ResultsForty-nine consecutive cases of invasive breast carcinoma were identified. Descriptive data regarding the MIB-1 counts, PhH3 counts, mitotic counts and IOD, separated by grade, are summarized in Table 2. Comparing the low grades (grade 1 and 2) and high grade (grade 3) shows that there is significantly more grade 3 tumors lying above the regression line (p = 0.017). Histological grades are identified with shade of grey (see legend).Full size imageVisual inspection of slides immunostained with PhH3 shows preparations with very high signal-to-noise ratio. Identification of mitoses is thus straightforward especially when compared with mitoses identification in H&E preparations.

Mitotic figures are counted at this magnification directly from the microscope.Full size imageDiscussionCell proliferation is an important component in the assessment of many neoplasms. This study evaluated the correlations between MIB-1, PhH3, and mitotic count in a defined area, focusing on invasive breast carcinoma. As expected, a very strong correlation (r = 0.94) between mitotic and PhH3 counts was found.
Given the optimization of our experimental design to reduce confounders in our comparisons, such as tumor heterogeneity, and the strong correlation between PhH3 and mitotic count found in this study, the discrepancy between MIB-1 and PhH3 or mitotic count would not be explained by experimental error. We suggest, based on review of the literature, that the discrepancy has pathological ground related to cell cycle anomalies found in malignant tumors.
A key factor would be the proportion of time a cell spends in the mitotic phase compared to interphase (G1-S-G2 phase). A careful examination of the results of this study indicates that the higher the tumor grade, the more likely that the tumor shows a higher MIB-1 count as compared to PhH3.
This is illustrated in Figure 2 which shows that the majority of low grade tumors lie below the linear regression while the majority of grade 3 tumors lie above the line.This non-linear relationship between grade and the MIB-1 to PhH3 ratio is a phenomenon that can be explained by increased relative durations spent in interphase as compared to mitotic phase in the higher grade tumors. High grade tumors generally have longer cell cycles and, in particular, longer S-phase durations (Ts).
This is because they are more likely to be aneuploid and thus more DNA material must be replicated46,47,48. In a study on DNA ploidy and S-phase fraction (SPF) in breast cancer by Wong et al., histologic grade 3 tumors were more frequently aneuploid and had higher SPF than grade 1 or 2 tumors46.
While the reason for increased SPF was not studied, a lengthened S-phase, due to increased DNA in aneuploid cells, is one of the most likely explanations. Similar findings are seen in studies regarding Ts seen in lung50, head and neck51, colon52,53, esophagus54 and cervix54.
These studies showed that aneuploid tumors displayed approximately 25–30% longer Ts than diploid tumors44. Additional explanations for lengthened interphase in higher grade tumors include increased damage to DNA replication machinery resulting in slower activity by enzymes such as DNA replicase, and increased malfunction of cell cycle control signals which would inhibit transition from interphase into mitosis. Moreover and importantly, while mitotic phase duration lengthens in malignant cells compared to benign cells, when considering only malignant cells, it remains relatively unaffected by ploidy number55,56. The proportion of time spent in the cell cycle but not in mitotic phase is thus generally higher in higher grade tumors: cell cycle phase durations are consequently decoupled. The consequences of this decoupled cell cycle phase duration can be modeled mathematically. The relationship between the number of cells in mitosis and number of cells in the cell cycle is represented by the equation where Nm is the number of cells undergoing mitosis at a given time, Nc is the number of cells in the cell cycle, Tm is the duration of mitosis, and Tc is the duration of cell cycle. Given that Tm is relatively constant, the proportion of cycling cell undergoing mitosis is inversely proportion to duration of the cell cycle. Since Nm can be estimated by the number of cells staining for PhH3, and Nc by the number cells staining for MIB-1, . This effect would explain “imperfect” correlation between PhH3 (or mitotic count) versus MIB-1. Also because of lengthened S phase two tumors with identical mitotic count could have different MIB-1 fractions.Full size imagePractically, two different tumors could have the same mitotic or PhH3 counts, but quite different MIB-1 counts. Some of the potential additional variables that are not accounted for by our model includes the G0 phase and the fact that MIB-1 does not perfectly stain all cells in the cell cycle, nor does PhH3 staining correspond precisely to mitosis.IOD results also corroborate with the above discussion.
The breast tumors with higher IOD would more likely be aneuploid and would also be associated with disproportionately longer S-phase.
Despite poor correlations with all proliferative markers, IOD did show the best correlation with MIB-1, evidence that nuclear content has stronger relationship to overall cell cycle duration than to mitotic duration. It is generally suggested that tumors with a higher proliferative fraction would proliferate faster.
However, the same proportional variation of P and Tc do not have the same impact in term of cell production. More than a proliferative or kinetic marker, these “P” markers, and especially MIB-1 since it captures the entire Tc, are probably better characterized as markers of cell-cycle abnormality.The previously reported correlations (Table 3), show significant variability between different reports and different tumor types. Part of this variability is explained by intratumoral heterogeneity which are not addressed by their methodologies; none of these studies directly compared proliferative markers on the same tumor areas. For example, in the study with the lowest correlations20, mitotic and MIB-1 counts were assessed from random fields in full sections while PhH3 was assessed on tissue micro-array (TMA).

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