Etiology of Leprosy:
A chronic infection caused by Mycobacterium leprae
Acid-fast, rod shaped
Main route of infection:
nasal droplets,
Eating armadillos (south america)
Not very contagious, but close relatives are at high risk of infection
Newer biomarkers,techniques & their inclusion in 2016 WHO classification for leukaemia/lymphomas increases the responsibility of the pathologists, requiring to develop an integrated multidisciplinary approach for reporting.
Etiology of Leprosy:
A chronic infection caused by Mycobacterium leprae
Acid-fast, rod shaped
Main route of infection:
nasal droplets,
Eating armadillos (south america)
Not very contagious, but close relatives are at high risk of infection
Newer biomarkers,techniques & their inclusion in 2016 WHO classification for leukaemia/lymphomas increases the responsibility of the pathologists, requiring to develop an integrated multidisciplinary approach for reporting.
By using flow cytometry, staining dyes are needed. Creative Bioarray can choose different dyes to perform the assays, including propidium iodide (PI), BrdU, 7-amino actinomycin-D (7-AAD), Hoechst 33342 and 33258, and 4’6’-diamidino-2-phenylindole (DAPI), based on the customer’s applications or requirements.
https://www.creative-bioarray.com/cell-cycle-assays.htm
Cell cycle refers to the set of events through which a cell grows, replicates its genome, and ultimately divides into two daughter cells through the process of mitosis.
https://www.creative-bioarray.com/cell-cycle-assays.htm
1. Lung Cancer
1
Men
Lung and bronchus 28%
Prostate 11%
Colon and rectum 8%
Pancreas 6%
Leukemia 4%
Women
Lung and bronchus 26%
Breast 15%
Colon and rectum 9%
Pancreas 7%
Ovary 6%
American Cancer Society. Cancer Facts & Figures 2011.
NSCLC
85%
Lung cancer subtypes
Squamous cell carcinoma 25%
Small-cell carcinoma 15%
Large-cell carcinoma 10%
Adenocarcinoma 45%
• Leading cause of cancer-related deaths
• Non-small cell lung cancer (NSCLC) is the most common form
3. Targets of LKB1 Kinase
3
• Cell polarity
• Proliferation
• CREB related gene
transcription
4. Knockdown of DTYMK in LKB1-WT and LKB1-
mutant NSCLC cell lines.
Liu Y et al. Cancer Discovery 2013;3:870-879
B
5. Aims
5
To investigate the effect of sensitivity of non-small cell
lung cancer cells expressing wild-type or mutant LKB1 to
inhibitors of CHK1/2 and NUAK1
Cell lines:
A549 (Human alveolar
adenocarcinoma cell line):
• A549LKB1- Intact LKB1
• A549 BABE- knockout LKB1
Compounds:
AZD7762 (CHK1/2 inhibitor])
WZ4002 (NUAK1 inhibitor)
6. What did we do?
Resazurin proliferation assay
- 1000 cells in 96 well plate; add dilution of drug, incubate 3 days, add resazurin, resazurin taken
up by living cells and reduced to fluorescent Resorufin
Western Blot
- Check for LKB1 presence/absence
FACS analysis
- Determine proportion of cells in G1 phase, high proportion suggests slow growth rate
PicoGreen proliferation assay
- 1000 cells into 96 well plate, for each day, remove cell medium, add lysis buffer, this fixes the
number of cells present on that day. PicoGreen dye + OMEGA plate reader.
12. PicoGreen assay
12
• The A549-LKB appeared to grow more rapidly
• The reduced DNA content on day 3 likely represents cells dying or becoming confluent
• AZD7762 has little effect in either cell line
• WZ4002 had a modest growth inhibitory effect in both cell lines
Fluorescence
Fluorescence
Days Days
13. Summary of results
13
1. LKB1 expression does not affect sensitivity to AZD7762 but increases sensitivity to
WZ4002 (slightly)
2. PARP levels lower in cells expressing LKB1, but uncertain relevance
3. Higher % of cells in G1 in LKB1 expressing cells
4. LKB1 expressing cells grew faster, but not differentially sensitive to AZD or WZ
5. Similar data in another cell line (A427)
6. More work needed to better understand results
Editor's Notes
LKB1 is an upstream kinase of AMPK
AMPK: many metabolic pathways
in general, switches off ATP consuming pathways and switched on ATP generating pathways.
AMPK activated by depleted ATP. ADP and AMP compete for binding to AMPK so ratios determine activation.
Activated by p in activation loop of kinase domain.
4 adenine binding clefts: 1 always unoccupies, 1 always bound AMP other 2 in competition. Presence of AMP/ADP promotes phosphorylation and inhibits dephosphorylation of Thr172
AMPK Related Kinases (ARKs)
Highly conserved sequence at the site of the Thr172
Play roles in: cell polarity, cell proliferation (NUAKs), CREB related gene transcription.
Knockdown of DTYMK in LKB1-WT and LKB1-mutant NSCLC cell lines. A, Western blot analyses of LKB1 expression in LKB1-WT (H358 and Calu-1) and LKB1-deficient (H2122 and A549) NSCLC cell lines. B, (shRNAs where used to knockdown DTYMK in LKB1 WT and LKB1-mutant cell lines. LKB1 mutant cell line show increased sensitivity to DTYMK knockdown)LKB1-WT (H358 and Calu-1) and LKB1-deficient (H2122 and A549) NSCLC cell lines were transduced with the indicated shRNAs for 1 day and then selected with 5 μg/mL puromycin (puro) for 2 days in six-well plates. The cells were collected by trypsin and replated into 96-well plates at 2,000 cells per well in 150 μL medium containing 5 μg/mL puromycin and measured daily using Promega's CellTiter-Glo Assay. The data represent mean ± SD for three replicates. C, the cells left from the replating were lysed for Western blot analysis of DTYMK expression. D, graph of dTDP levels in A549 cells transduced with the indicated shRNA for 4 days. The data represent mean ± SD for six replicates. Expression of DTYMK in these cells at the time of metabolite extraction was determined by Western blotting. E, LKB1-WT (H358 and Calu-1) and LKB1-deficient (H2122 and A549) NSCLC cell lines were plated into 96-well plates at 2,000 cells per well in 150 μL medium containing the indicated concentrations of AZD7762 or CHIR124 for 3 days. Viable cells were counted daily using Dojindo's Cell Counting Kit-8 Assay. The data represent mean ± SD for three repeats. Bottom: GI50 was calculated with GraphPad. (LKB1 Defficient cells are more sensitive to Chk1 inhibitors that LKB1 WT cells. Suggest more DNA damage in LKB1 mutated cells)
The results show that both the cell lines (A549 LKB1 and A549 BABE) show a decrease in relative resazurin fluorescence at the highest concentration of the drug WZ4002. Only once the concentration of WZ4002 reaches 3 µM does the resazurin fluorescence begin to decrease. At lower concentrations than this WZ4002 has no obvious effect on the cells.
AZD7762 has a more severe effect on the cells than WZ4002, as the proliferation of the cells is effectively decreased at concentrations as low as 0.03. At 0.3 µM AZD7762 cell growth is reduced by around 90%. At 10µM AZD6672 is extremely toxic to cell growth, the cells showing almost no proliferation at all. Both cell lines (A549 BABE and A549 LKB1) are effected to a similar extent by AZD7762.
These results give us an indication of the concentrations of each drug at which we can expect to see drug response. We decided to run a PicoGreen assay treating cells with 0.1uM AZD and 3uM WZ.
We used western blot to test both quantitatively and qualitatively for the presence of the proteins LKB1, the DNA damage response protein PARP, and for actin to use as a control. These proteins were probed for in both the A549 BABE and A549 LKB cells, either untreated, or treated with either the AZD7762 drug or the WZ40092 drug.
From the western blot the abundance of each of the two proteins (PARP & LKB1) was calculated relative to the amount of actin. As expected LKB1 was not present in the BABE cells, but was present in the LKB1 cells. PARP was present at higher levels in the BABE cells, suggesting that the absense of LKB1 may be contributing to higher levels of DNA damage and therefore increased expression of the DNA damage response gene Parp.
FACS analysis was used to investigate the cell cycle of the cells,
Results from the PicoGreen assay of the A549 BABE cells. Control cells shown in blue. The results show that the addition of 0.1uMAZD does not have much of an affect on the cells. They display a slightly slower rate of proliferation between day 0 and day 1 however this is quickly overcome and the cells show a higher proliferation rate than the control cells. Addition of 3uM WZ however has a pronounced negative effect on the proliferation of the cells, displaying only roughly half the fluorescence intensity as the AZD treated cells on day 2.
