Generating New FANCA-Deficient HNSCC Cell Lines by Genomic Editing Recapitulate the Cellular Phenotypes of Fanconi Anemia

Fanconi anemia (FA) patients have an exacerbated risk of head and neck squamous cell carcinoma (HNSCC). Treatment is challenging as FA patients display enhanced toxicity to standard treatments, including radio/chemotherapy. Therefore better therapies as well as new disease models are urgently needed. We have used CRISPR/Cas9 editing tools in order to interrupt the human FANCA gene by the generation of insertions/deletions (indels) in exon 4 in two cancer cell lines from sporadic HNSCC having no mutation in FA-genes: CAL27 and CAL33 cells. Our approach allowed efficient editing, subsequent purification of single-cell clones, and Sanger sequencing validation at the edited locus. Clones having frameshift indels in homozygosis did not express FANCA protein and were selected for further analysis. When compared with parental CAL27 and CAL33, FANCA-mutant cell clones displayed a FA-phenotype as they i) are highly sensitive to DNA interstrand crosslink (ICL) agents such as mitomycin C (MMC) or cisplatin, ii) do not monoubiquitinate FANCD2 upon MMC treatment and therefore iii) do not form FANCD2 nuclear foci, and iv) they display increased chromosome fragility and G2 arrest after diepoxybutane (DEB) treatment. These FANCA-mutant clones display similar growth rates as their parental cells. Interestingly, mutant cells acquire phenotypes associated with more aggressive disease, such as increased migration in wound healing assays. Therefore, CAL27 and CAL33 cells with FANCA mutations are phenocopies of FA-HNSCC cells.

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Generating new FANCA-deficient HNSCC cell lines by genomic editing recapitulate the cellular phenotypes of Fanconi anemia

10.1101/2020.10.03.324921 ◽

2020 ◽

Author(s):

Ricardo Errazquin ◽

Esther Sieiro ◽

Pilar Moreno ◽

María José Ramirez ◽

Corina Lorz ◽

...

Keyword(s):

Cell Lines ◽

Fanconi Anemia ◽

Mutant Cell ◽

Interstrand Crosslink ◽

Cell Clones ◽

Highly Sensitive ◽

Chromosome Fragility ◽

Genomic Editing ◽

Cellular Phenotypes ◽

Radio Chemotherapy

AbstractFanconi anemia (FA) patients have an exacerbated risk of head and neck squamous cell carcinoma (HNSCC). Treatment is challenging as FA patients display enhanced toxicity to standard treatments, including radio/chemotherapy. Therefore better therapies as well as new disease models are urgently needed. We have used CRISPR/Cas9 editing tools in order to interrupt the human FANCA gene by the generation of insertions/deletions (indels) in exon 4 in two cancer cell lines from sporadic HNSCC having no mutation in FA-genes: CAL27 and CAL33 cells. Our approach allowed efficient editing, subsequent purification of single-cell clones, and Sanger sequencing validation at the edited locus. Clones having frameshift indels in homozygosis did not express FANCA protein and were selected for further analysis. When compared with parental CAL27 and CAL33, FANCA-mutant cell clones displayed a FA-phenotype as they i) are highly sensitive to DNA interstrand crosslink (ICL) agents such as mitomycin C (MMC) or cisplatin, ii) do not monoubiquitinate FANCD2 upon MMC treatment and therefore iii) do not form FANCD2 nuclear foci, and iv) they display increased chromosome fragility and G2 arrest after diepoxybutane (DEB) treatment. These FANCA-mutant clones display similar growth rates as their parental cells. Interestingly, mutant cells acquire phenotypes associated with more aggressive disease, such as increased migration in wound healing assays. Therefore, CAL27 and CAL33 cells with FANCA mutations are phenocopies of FA-HNSCC cells.

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Generating New FANCA-Deficient HNSCC Cell Lines by Genomic Editing Recapitulates the Cellular Phenotypes of Fanconi Anemia

Genes ◽

10.3390/genes12040548 ◽

2021 ◽

Vol 12 (4) ◽

pp. 548

Author(s):

Ricardo Errazquin ◽

Esther Sieiro ◽

Pilar Moreno ◽

María José Ramirez ◽

Corina Lorz ◽

...

Keyword(s):

Cell Lines ◽

Fanconi Anemia ◽

Mutant Cell ◽

Interstrand Crosslink ◽

Cell Clones ◽

Highly Sensitive ◽

Chromosome Fragility ◽

Genomic Editing ◽

Cellular Phenotypes ◽

Radio Chemotherapy

Fanconi anemia (FA) patients have an exacerbated risk of head and neck squamous cell carcinoma (HNSCC). Treatment is challenging as FA patients display enhanced toxicity to standard treatments, including radio/chemotherapy. Therefore, better therapies as well as new disease models are urgently needed. We have used CRISPR/Cas9 editing tools in order to interrupt the human FANCA gene by the generation of insertions/deletions (indels) in exon 4 in two cancer cell lines from sporadic HNSCC having no mutation in FA-genes: CAL27 and CAL33 cells. Our approach allowed efficient editing, subsequent purification of single-cell clones, and Sanger sequencing validation at the edited locus. Clones having frameshift indels in homozygosis did not express FANCA protein and were selected for further analysis. When compared with parental CAL27 and CAL33, FANCA-mutant cell clones displayed a FA-phenotype as they (i) are highly sensitive to DNA interstrand crosslink (ICL) agents such as mitomycin C (MMC) or cisplatin, (ii) do not monoubiquitinate FANCD2 upon MMC treatment and therefore (iii) do not form FANCD2 nuclear foci, and (iv) they display increased chromosome fragility and G2 arrest after diepoxybutane (DEB) treatment. These FANCA-mutant clones display similar growth rates as their parental cells. Interestingly, mutant cells acquire phenotypes associated with more aggressive disease, such as increased migration in wound healing assays. Therefore, CAL27 and CAL33 cells with FANCA mutations are phenocopies of FA-HNSCC cells.

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On the role of FAN1 in Fanconi anemia

Blood ◽

10.1182/blood-2012-04-420604 ◽

2012 ◽

Vol 120 (1) ◽

pp. 86-89 ◽

Cited By ~ 22

Author(s):

Juan P. Trujillo ◽

Leonardo B. Mina ◽

Roser Pujol ◽

Massimo Bogliolo ◽

Joris Andrieux ◽

...

Keyword(s):

Fanconi Anemia ◽

Bone Marrow Failure ◽

Pcr Amplification ◽

Minor Role ◽

Interstrand Crosslink ◽

Genetic Features ◽

Chromosome Fragility ◽

A Minor ◽

Interstrand Crosslink Repair ◽

Crosslink Repair

Abstract Fanconi anemia (FA) is a rare bone marrow failure disorder with defective DNA interstrand crosslink repair. Still, there are FA patients without mutations in any of the 15 genes individually underlying the disease. A candidate protein for those patients, FA nuclease 1 (FAN1), whose gene is located at chromosome 15q13.3, is recruited to stalled replication forks by binding to monoubiquitinated FANCD2 and is required for interstrand crosslink repair, suggesting that mutation of FAN1 may cause FA. Here we studied clinical, cellular, and genetic features in 4 patients carrying a homozygous 15q13.3 micro-deletion, including FAN1 and 6 additional genes. Biallelic deletion of the entire FAN1 gene was confirmed by failure of 3′- and 5′-PCR amplification. Western blot analysis failed to show FAN1 protein in the patients' cell lines. Chromosome fragility was normal in all 4 FAN1-deficient patients, although their cells showed mild sensitivity to mitomycin C in terms of cell survival and G2 phase arrest, dissimilar in degree to FA cells. Clinically, there were no symptoms pointing the way to FA. Our results suggest that FAN1 has a minor role in interstrand crosslink repair compared with true FA genes and exclude FAN1 as a novel FA gene.

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FANCN Hypomorphic Mutation Retains BRCA1 Binding Domain

Blood ◽

10.1182/blood.v128.22.2676.2676 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2676-2676

Author(s):

Elizabeth Thompson ◽

Jakub Tolar ◽

Eric A. Hendrickson

Keyword(s):

Bone Marrow ◽

Cell Line ◽

Gene Targeting ◽

Cell Lines ◽

Fanconi Anemia ◽

Bone Marrow Failure ◽

Mutant Cell ◽

Genomic Stability ◽

Binding Domain ◽

Essential Function

Abstract Introduction: Fanconi anemia (FA) is a recessive genetic disease that can result in bone marrow failure, morphological defects and cancer predisposition. Patients with mutations in the FA gene, FANCN/PALB2 (partner and localizer of BRCA2) typically have more severe disease with earlier onset of cancer and bone marrow failure. FANCN is known to promote homologous recombination (HR) and contains an N-terminal BRCA1 binding domain, and a C-terminal BRCA2 binding domain. To further investigate the function of FANCN, we used recombinant adeno-associated virus (rAAV)-mediated gene targeting techniques to make a conditional null FANCN cell line. We targeted early exons 2 and 3 and found that removal of the conditional allele resulted in spontaneous chromosomal breaks and rearrangements that ultimately lead to chromosomal catastrophe within 48 hours. Due to the essential function of FANCN in genome stability, we were curious how FANCN patients can survive with biallelic loss of function mutations in FANCN. Methods: A hypomorphic FANCN cell line was created using clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR associated 9 (Cas9)-mediated gene targeting of FANCN in exon 5. In addition, we complemented the FANCN conditional null cell line with four different FANCN cDNAs containing patient mutations with premature stop codons that completely eliminate or disrupt the N-terminal BRCA2 binding domain. These FANCN mutant cell lines have been investigated for expression of truncated protein and will be tested in their ability to bind and form foci with BRCA1 and BRCA2. Finally, these FANCN mutant cell lines will be evaluated in their sensitivity to mitomycin C (MMC), PARP inhibitors, replication stress, and in their ability to repair DNA. Results: We have determined that FANCN is essential for maintaining genomic stability and cellular viability. However, contrary to the lethality of deleting exons 2 and 3, the biallelic frameshift mutations in exon 5 resulted in a viable FANCN mutant cell line. These hypomorphic FANCN exon 5 frameshift mutations truncate the C-terminal BRCA2 binding domain, and this cell line has a growth defect and MMC sensitivity. Furthermore, we have demonstrated that the patient mutation complemented cell lines are expressing truncated FANCN proteins that will be functionally characterized. Conclusions: We are beginning to unveil important and separate functional domains of FANCN. For example, the FANCN N-terminus with the BRCA1 binding domain serves an essential function in viability and genomic stability, whereas the FANCN C-terminus with the BRCA2 binding domain servers an essential role in cell proliferation and MMC resistance. This research is expected to continue to advance our mechanistic understanding of FANCN and how these FANCN patient mutations result in disease. In addition, these novel FANCN cell lines provide a means for screening potential new therapeutics for the treatment of Fanconi anemia. Disclosures Hendrickson: Horizon Discovery , Humanetics and Intellia Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: licensing fees, Research Funding.

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BHK-21 Cell Clones Differ in Chikungunya Virus Infection and MXRA8 Receptor Expression

Viruses ◽

10.3390/v13060949 ◽

2021 ◽

Vol 13 (6) ◽

pp. 949

Author(s):

Peiqi Yin ◽

Margaret Kielian

Keyword(s):

Cell Lines ◽

Primary Infection ◽

Sindbis Virus ◽

Chikungunya Virus ◽

Mrna Level ◽

Receptor Expression ◽

Chikv Infection ◽

Cell Clones ◽

Clonal Cell Lines ◽

Exogenous Expression

Baby hamster kidney-21 (BHK-21) cells are widely used to propagate and study many animal viruses using infection and transfection techniques. Among various BHK-21 cell clones, the fibroblast-like BHK-21/C-13 line and the epithelial-like BHK-21/WI-2 line are commonly used cell clones for alphavirus research. Here we report that BHK-21/WI-2 cells were significantly less susceptible to primary infection by the alphavirus chikungunya virus (CHIKV) than were BHK-21/C-13 cells. The electroporation efficiency of alphavirus RNA into BHK-21/WI-2 was also lower than that of BHK-21/C-13. The growth of CHIKV was decreased in BHK-21/WI-2 compared to BHK-21/C-13, while primary infection and growth of the alphavirus Sindbis virus (SINV) were equivalent in the two cell lines. Our results suggested that CHIKV entry could be compromised in BHK-21/WI-2. Indeed, we found that the mRNA level of the CHIKV receptor MXRA8 in BHK-21/WI-2 cells was much lower than that in BHK-21/C-13 cells, and exogenous expression of either human MXRA8 or hamster MXRA8 rescued CHIKV infection. Our results affirm the importance of the MXRA8 receptor for CHIKV infection, and document differences in its expression in two clonal cell lines derived from the original BHK-21 cell cultures. Our results also indicate that CHIKV propagation and entry studies in BHK-21 cells will be significantly more efficient in BHK-21/C-13 than in BHK-21/WI-2 cells.

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DDRE-03. IDH1-MUTANT GBM CELLS ARE HIGHLY SENSITIVE TO COMBINATION OF KDM6A/B AND HDAC INHIBITORS

Neuro-Oncology Advances ◽

10.1093/noajnl/vdab024.025 ◽

2020 ◽

Vol 3 (Supplement_1) ◽

pp. i6-i7

Author(s):

Alişan Kayabölen ◽

Gizem Nur Sahin ◽

Fidan Seker ◽

Ahmet Cingöz ◽

Bekir Isik ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

Cell Lines ◽

Mutant Cell ◽

Glioma Cells ◽

Epigenetic Therapy ◽

Low Grade ◽

Orthotopic Model ◽

Cycle Arrest ◽

Mutant Cells

Abstract Mutations in IDH1 and IDH2 genes are common in low grade gliomas and secondary GBM and are known to cause a distinct epigenetic landscape in these tumors. To interrogate the epigenetic vulnerabilities of IDH-mutant gliomas, we performed a chemical screen with inhibitors of chromatin modifiers and identified 5-azacytidine, Chaetocin, GSK-J4 and Belinostat as potent agents against primary IDH1-mutant cell lines. Testing the combinatorial efficacy of these agents, we demonstrated GSK-J4 and Belinostat combination as a very effective treatment for the IDH1-mutant glioma cells. Engineering established cell lines to ectopically express IDH1R132H, we showed that IDH1R132H cells adopted a different transcriptome with changes in stress-related pathways that were reversible with the mutant IDH1 inhibitor, GSK864. The combination of GSK-J4 and Belinostat was highly effective on IDH1R132H cells, but not on wt glioma cells or nonmalignant fibroblasts and astrocytes. The cell death induced by GSK-J4 and Belinostat combination involved the induction of cell cycle arrest and apoptosis. RNA sequencing analyses revealed activation of inflammatory and unfolded protein response pathways in IDH1-mutant cells upon treatment with GSK-J4 and Belinostat conferring increased stress to glioma cells. Specifically, GSK-J4 induced ATF4-mediated integrated stress response and Belinostat induced cell cycle arrest in primary IDH1-mutant glioma cells; which were accompanied by DDIT3/CHOP-dependent upregulation of apoptosis. Moreover, to dissect out the responsible target histone demethylase, we undertook genetic approach and demonstrated that CRISPR/Cas9 mediated ablation of both KDM6A and KDM6B genes phenocopied the effects of GSK-J4 in IDH1-mutant cells. Finally, GSK-J4 and Belinostat combination significantly decreased tumor growth and increased survival in an orthotopic model in mice. Together, these results suggest a potential combination epigenetic therapy against IDH1-mutant gliomas.

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Detection of somatic mosaicism and classification of Fanconi anemia patients by analysis of the FA/BRCA pathway

Blood ◽

10.1182/blood-2004-05-1852 ◽

2005 ◽

Vol 105 (3) ◽

pp. 1329-1336 ◽

Cited By ~ 78

Author(s):

Jean Soulier ◽

Thierry Leblanc ◽

Jérôme Larghero ◽

Hélène Dastot ◽

Akiko Shimamura ◽

...

Keyword(s):

Fanconi Anemia ◽

Cancer Susceptibility ◽

Bone Marrow Failure ◽

Somatic Mosaicism ◽

Chromosome Breakage ◽

Peripheral Blood Lymphocytes ◽

Specific Analysis ◽

Number Of Patients ◽

Chromosome Fragility

AbstractFanconi anemia (FA) is characterized by congenital abnormalities, bone marrow failure, chromosome fragility, and cancer susceptibility. Eight FA-associated genes have been identified so far, the products of which function in the FA/BRCA pathway. A key event in the pathway is the monoubiquitination of the FANCD2 protein, which depends on a multiprotein FA core complex. In a number of patients, spontaneous genetic reversion can correct FA mutations, leading to somatic mosaicism. We analyzed the FA/BRCA pathway in 53 FA patients by FANCD2 immunoblots and chromosome breakage tests. Strikingly, FANCD2 monoubiquitination was detected in peripheral blood lymphocytes (PBLs) in 8 (15%) patients. FA reversion was further shown in these patients by comparison of primary fibro-blasts and PBLs. Reversion was associated with higher blood counts and clinical stability or improvement. Once constitutional FANCD2 patterns were determined, patients could be classified based on the level of FA/BRCA pathway disruption, as “FA core” (upstream inactivation; n = 47, 89%), FA-D2 (n = 4, 8%), and an unidentified downstream group (n = 2, 4%). FA-D2 and unidentified group patients were therefore relatively common, and they had more severe congenital phenotypes. These results show that specific analysis of the FA/BRCA pathway, combined with clinical and chromosome breakage data, allows a comprehensive characterization of FA patients.

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Tissue type-specific expression of intermediate filament proteins in a cultured epithelial cell line from bovine mammary gland

The Journal of Cell Biology ◽

10.1083/jcb.96.1.37 ◽

1983 ◽

Vol 96 (1) ◽

pp. 37-50 ◽

Cited By ~ 93

Author(s):

E Schmid ◽

DL Schiller ◽

C Grund ◽

J Stadler ◽

WW Franke

Keyword(s):

Mammary Gland ◽

Epithelial Cell ◽

Cell Line ◽

Cell Lines ◽

Intermediate Filament ◽

Striking Difference ◽

Intermediate Filament Proteins ◽

Cell Clones ◽

Vimentin Filaments

Different clonal cell lines have been isolated from cultures of mammary gland epithelium of lactating cow's udder and have been grown in culture media containing high concentrations of hydrocortisone, insulin, and prolactin. These cell (BMGE+H), which grow in monolayers of typical epithelial appearance, are not tightly packed, but leave intercellular spaces spanned by desmosomal bridges. The cells contain extended arrays of cytokeratin fibrils, arranged in bundles attached to desmosomes. Gel electophoresis show that they synthesize cytokeratins similar, if not identical, to those found in bovine epidermis and udder, including two large (mol wt 58,500 and 59,000) and basic (pH range: 7-8) and two small (mol wt 45,500 and 50,000) and acidic (pH 5.32 and 5.36) components that also occur in phosphorylated forms. Two further cytokeratins of mol wts 44,000 (approximately pH 5.7) and 53,000 (pH 6.3) are detected as minor cytokeratins in some cell clones. BMGE+H cells do not produce vimentin filaments as determined by immunofluorescence microscopy and gel electrophoresis. By contrast, BMGE-H cells, which have emerged from the same original culture but have been grown without hormones added, are not only morphologically different, but also contain vimentin filaments and a different set of cytokeratins, the most striking difference being the absence of the two acidic cytokeratins of mol wt 50,000 and 45,500. Cells of the BMGE+H line are characterized by an unusual epithelial morphology and represent the first example of a nonmalignant permanent cell line in vitro that produces cytokeratin but not vimentin filaments. The results show that (a) tissue-specific patterns of intermediate filament expression can be maintained in permanent epithelial cell lines in culture, at least under certain growth conditions; (b) loss of expression of relatively large, basic cytokeratins is not an inevitable consequence of growth of epithelial cells in vitro. Our results further show that, during culturing, different cell clones with different cytoskeletal composition can emerge from the same cell population and suggest that the presence of certain hormones may have an influence on the expression of intermediate filament proteins.

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Functional analysis of regulatory defects in HLA class II negative mutant cell lines

Human Immunology ◽

10.1016/0198-8859(89)90811-2 ◽

1989 ◽

Vol 26 ◽

pp. 113

Author(s):

Linda Shookster ◽

Clifford R Hume ◽

Janet S Lee

Keyword(s):

Functional Analysis ◽

Cell Lines ◽

Mutant Cell ◽

Class Ii ◽

Hla Class Ii ◽

Negative Mutant

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Isolation of Cell Lines That Show Novel, Murine Leukemia Virus-Specific Blocks to Early Steps of Retroviral Replication

Journal of Virology ◽

10.1128/jvi.79.20.12969-12978.2005 ◽

2005 ◽

Vol 79 (20) ◽

pp. 12969-12978 ◽

Cited By ~ 16

Author(s):

James W. Bruce ◽

Kenneth A. Bradley ◽

Paul Ahlquist ◽

John A. T. Young

Keyword(s):

Cell Lines ◽

Reverse Transcription ◽

Murine Leukemia Virus ◽

Leukemia Virus ◽

Mutant Cell ◽

Chinese Hamster ◽

High Volume ◽

Early Stages ◽

Retroviral Replication ◽

Murine Leukemia

ABSTRACT In order to identify cellular proteins required for early stages of retroviral replication, a high volume screening with mammalian somatic cells was performed. Ten pools of chemically mutagenized Chinese hamster ovary (CHO-K1) cells were challenged with a murine leukemia virus (MLV) vector pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G), and cells that failed to be transduced were enriched by cell sorting. Each pool yielded a clonally derived cell line with a 5-fold or greater resistance to virus infection, and five cell lines exhibited a >50-fold resistance. These five cell lines were efficiently infected by a human immunodeficiency virus vector pseudotyped with VSV-G. When engineered to express the TVA receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A), the five cell lines were resistant to infection with a MLV vector pseudotyped with the ASLV-A envelope protein but were fully susceptible to infection with an ASLV-A vector. Thus, the defect in these cells resides after virus-cell membrane fusion and, unlike those in other mutant cell lines that have been described, is specific for the MLV core. To identify the specific stages of MLV infection that are impaired in the resistant cell lines, real-time quantitative PCR analyses were employed and two phenotypic groups were identified. Viral infection of three cell lines was restricted before reverse transcription; in the other two cell lines, it was blocked after reverse transcription, nuclear localization, and two-long terminal repeat circle formation but before integration. These data provide genetic evidence that at least two distinct intracellular gene products are required specifically for MLV infection. These cell lines are important tools for the biochemical and genetic analysis of early stages in retrovirus infection.

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