Improvement of the BALB/c-3T3 cell transformation assay: a tool for investigating cancer mechanisms and therapies

Poburski, Doerte; Thierbach, René

doi:10.1038/srep32966

Cited by 24 publications

(24 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In our previous publication we already demonstrated how the BALB-CTA can be successfully combined with several endpoint applications to be used for mechanistic cancer research3. To study the dysregulation of an insulin/IGF-1 signaling we combined OSI-906 with the two-stage BALB-CTA.…”

Section: Discussionmentioning

confidence: 99%

“…A two-stage BALB-CTA was performed as previously described3. In detail, 5000 cells per well were seeded as 4 replicates into Corning® Primaria™ 6-well plates (VWR #734-0077) and cultured under standard conditions (37 °C, 5% CO 2 , 95% humidity) for 42 days, to obtain sufficient foci formation.…”

Section: Methodsmentioning

confidence: 99%

“…Basic knowledge of alterations in cellular neoplastic processes can be obtained with in vitro cell transformation assays (CTAs)3. CTAs mimic different stages of in vivo carcinogenesis and represent an excellent alternative to study cancer mechanisms and therapeutic options4.…”

mentioning

confidence: 99%

“…Our group further improved the BALB-CTA and combined the standard protocol with a parallel treatment of substances of interest. In addition, the BALB-CTA was combined with several endpoint applications, like analysis of protein level and signaling (western blot, immunofluorescence, subcellular fractionation) as well as parameters of energy metabolism (glucose and oxygen consumption)3. Hence, the BALB-CTA is more than a standard toxicological method and can provide essential information regarding key proteins and their signaling during the different stages of cell transformation and can help to identify potential cancer therapeutics.…”

mentioning

confidence: 99%

See 3 more Smart Citations

Insulin-IGF signaling affects cell transformation in the BALB/c 3T3 cell model

Poburski

Leovsky

Boerner

et al. 2016

Sci Rep

Self Cite

View full text Add to dashboard Cite

The increased cancer mortality of diabetes type 2 patients is most likely an evidence of the tight connection between tumor development and energy metabolism. A major focus of today’s research is still the identification of key proteins of both diseases and the development of corresponding inhibitors. In this study we combined the two-stage BALB/c-3T3 cell transformation assay (BALB-CTA) with the IR/IGF-1R inhibitor OSI-906 (linsitinib) and analyzed alterations in protein activity and energy parameters in non-transformed as well as transformed cells. OSI-906 successfully inhibited the phosphorylation of IR/IGF-1R and decreased cell growth in non-transformed cells. In the BALB-CTA, a permanent treatment with OSI-906 reduced cellular transformation dose-dependently, whereas a temporary treatment gave evidence for a preventive effect in the promotion phase. Furthermore, even though several key proteins were affected, it was possible to show that the phosphorylation of GSK3, Erk 1/2 and the S6 protein are not crucial for the cell foci reducing effect of OSI-906. Taken together, the BALB-CTA confirmed results of OSI-906 from animal studies and enhanced the knowledge of its mode of action. Therefore, the BALB-CTA offers the opportunity to analyze alterations in the transformation process more precisely and will be helpful to identify effective cancer treatments.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Insulin-IGF signaling affects cell transformation in the BALB/c 3T3 cell model

Poburski

Leovsky

Boerner

et al. 2016

Sci Rep

Self Cite

View full text Add to dashboard Cite

show abstract

“…Recently, the importance of a mechanism‐based approach in the risk assessment of nongenotoxic carcinogens has increased because nongenotoxic carcinogens act through a variety of specific mechanisms. Poburski and Thierbach [] combined the BALB/c cell transformation assay with several endpoint applications for protein analysis or energy parameter measurements to elucidate cancer mechanisms in more detail. Other laboratories have also reported integrated approach including the 3 T3 CTA and whole‐genome transcriptomic profiles [Vaccari et al ; Chang et al ].…”

Section: Introductionmentioning

confidence: 99%

Genome‐wide DNA methylation changes in transformed foci induced by nongenotoxic carcinogens

Hwang

Yeom

Eom

et al. 2019

Environ and Mol Mutagen

View full text Add to dashboard Cite

In vitro cell transformation assays (CTA) have been proposed as a method to identify possible nongenotoxic carcinogens. However, the current protocols do not provide information on the mechanism of action of the test articles. In this study, we combined an in vitro Bhas 42 CTA and sequencing‐based DNA methylation profiling analysis to elucidate the carcinogenic mechanism associated with nongenotoxic carcinogens. Three nongenotoxic carcinogens were evaluated: cadmium chloride, methyl carbamate, and lithocholic acid. Methylation profiles were generated for the two nongenotoxic carcinogens (cadmium chloride and lithocholic acid) that were positive in Bhas 42 CTA. Methyl carbamate did not exhibit any promoter activity. Approximately 9.8% of all differentially methylated regions (DMRs) identified in cadmium chloride‐induced transformed foci overlapped with DMRs in lithocholic acid‐induced transformed foci. Interestingly, overlapping DMRs showed more hypermethylation than individual DMRs. In addition, the DMRs in CpG island elements common to both nongenotoxic carcinogens showed considerably more bias toward hypermethylated DMRs than those unique to either cadmium chloride or lithocholic acid. Pathway enrichment analysis revealed that genes harboring hypermethylated DMRs were significantly enriched in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways including pathways in cancer, basal cell carcinoma, and Wnt signaling. The genes harboring hypomethylated DMRs were significantly related to mRNA surveillance pathway, RNA transport, and autophagy. Taken together, our preliminary results on genome‐wide methylation analysis of cell clones from nongenotoxic carcinogen‐induced foci could be exploited for CTAs improvement, but further research will be required to standardize and assess the specificity and sensitivity of this combined approach. Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc.

show abstract

Mechanistic insights into p53‐regulated cytotoxicity of combined entinostat and irinotecan against colorectal cancer cells

et al. 2021

Self Cite

View full text Add to dashboard Cite

Late-stage colorectal cancer (CRC) is still a clinically challenging problem. The activity of the tumor suppressor p53 is regulated via posttranslational modifications (PTMs). While the relevance of p53 Cterminal acetylation for transcriptional regulation is well-defined, it is unknown whether this PTM controls mitochondrially mediated apoptosis directly. We used wild-type p53 or p53-negative human CRC cells, cells with acetylation-defective p53, transformation assays, CRC organoids, and xenograft mouse models to assess how p53 acetylation determines cellular stress responses. The topoisomerase-1 inhibitor irinotecan induces acetylation of several lysine residues within p53. Inhibition of histone deacetylaces (HDACs) with the class I HDAC inhibitor entinostat synergistically triggers mitochondrial damage and apoptosis in irinotecan-treated p53-positive CRC cells. This specifically relies on the C-terminal acetylation of p53 by CREB binding protein (CBP)/p300 and the presence of C-terminally acetylated p53 in complex with the pro-apoptotic BCL2 antagonist/killer (BAK) protein. This control of C-terminal acetylation by HDACs can mechanistically explain why combinations of irinotecan and entinostat represent clinically tractable agents for the therapy of p53-proficient CRC.

show abstract

Improvement of the BALB/c-3T3 cell transformation assay: a tool for investigating cancer mechanisms and therapies

Cited by 24 publications

References 38 publications

Insulin-IGF signaling affects cell transformation in the BALB/c 3T3 cell model

Insulin-IGF signaling affects cell transformation in the BALB/c 3T3 cell model

Genome‐wide DNA methylation changes in transformed foci induced by nongenotoxic carcinogens

Mechanistic insights into p53‐regulated cytotoxicity of combined entinostat and irinotecan against colorectal cancer cells

Contact Info

Product

Resources

About