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Cytogenetic, Genomic, and Functional Characterization of Pituitary Gonadotrope Cell Lines.

ABSTRACT: L?T2 and ?T3-1 are important, widely studied cell line models for the pituitary gonadotropes that were generated by targeted tumorigenesis in transgenic mice. L?T2 cells are more mature gonadotrope precursors than ?T3-1 cells. Microsatellite authentication patterns, chromosomal characteristics, and their intercellular variation have not been reported. We performed microsatellite and cytogenetic analysis of both cell types at early passage numbers. Short tandem repeat (STR) profiling was consistent with a mixed C57BL/6J × BALB/cJ genetic background, with distinct patterns for each cell type. Spectral karyotyping in ?T3-1 cells revealed cell-to-cell variation in chromosome composition and pseudodiploidy. In L?T2 cells, chromosome counting and karyotyping demonstrated pseudotriploidy and high chromosomal variation among cells. Chromosome copy number variation was confirmed by single-cell DNA sequencing. Chromosomal compositions were consistent with a male sex for ?T3-1 and a female sex for L?T2 cells. Among L?T2 stocks used in multiple laboratories, we detected two genetically similar but distinguishable lines via STR authentication, L?T2a and L?T2b. The two lines differed in morphological appearance, with L?T2a having significantly smaller cell and nucleus areas. Analysis of immediate early gene and gonadotropin subunit gene expression revealed variations in basal expression and responses to continuous and pulsatile GnRH stimulation. L?T2a showed higher basal levels of Egr1, Fos, and Lhb but lower Fos induction. Fshb induction reached significance only in L?T2b cells. Our study highlights the heterogeneity in gonadotrope cell line genomes and provides reference STR authentication patterns that can be monitored to improve experimental reproducibility and facilitate comparisons of results within and across laboratories.

SUBMITTER: Ruf-Zamojski F

PROVIDER: S-EPMC6469952 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Cytogenetic, Genomic, and Functional Characterization of Pituitary Gonadotrope Cell Lines.

Ruf-Zamojski Frederique F Ge Yongchao Y Pincas Hanna H Shan Jidong J Song Yinghui Y Hines Nika N Kelley Kevin K Montagna Cristina C Nair Pranav P Toufaily Chirine C Bernard Daniel J DJ Mellon Pamela L PL Nair Venugopalan V Turgeon Judith L JL Sealfon Stuart C SC

Journal of the Endocrine Society 20190325 5

LβT2 and αT3-1 are important, widely studied cell line models for the pituitary gonadotropes that were generated by targeted tumorigenesis in transgenic mice. LβT2 cells are more mature gonadotrope precursors than αT3-1 cells. Microsatellite authentication patterns, chromosomal characteristics, and their intercellular variation have not been reported. We performed microsatellite and cytogenetic analysis of both cell types at early passage numbers. Short tandem repeat ...[more]

PMID: 31020055

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Project description:Gonadotrope or null cell pituitary tumors present clinically with signs of hypogonadism and hypopituitarism, together with visual disturbances due to mass effects. Since there are no medical therapies, surgery and/or radiation are the only therapeutic options. To identify dysregulated genes and/or pathways that may play a role in tumorigenesis and/ or progression, molecular profiling was performed on 14 gonadotrope tumors and 9 normal human pituitaries from autopsy samples. Principle component analysis (PCA) revealed clear discrimination between tumor and normal pituitary gene expression profiles. Bioinformatic analysis identified specific genes and pathways that were highly differentially regulated, including a cohort of putative downstream effectors of p53 were repressed in gonadotrope pituitary tumors, including GADD45β, GADD45γ and Reprimo with concomitant downregulation of the upstream regulator, PLAGL1. PLAGL1 reexpression in gonadotrope cells did not directly modulate the downstream targets. Further functional analysis of GADD45β was performed. Overexpression of GADD45β in mouse gonadotrope cells blocked proliferation, increased rates of apoptosis in response to growth factor withdrawal and increased colony formation in soft agar. In contrast to prior studies with GADD45γ, methylation interference assays showed no evidence of epigenetic modification of the GADD45β promoter in pituitary tumors. Thus, our data suggest that many components downstream of p53 are suppressed in gonadotrope pituitary tumors. A novel candidate, GADD45β is low in tumors and reexpression blocks proliferation, survival and tumorigenesis in gonadotrope cells. Unlike GADD45γ, GADD45β is not methylated to block its expression. Together these studies identify new targets and mechanisms to explore concerning pituitary tumor initiation and progression. To elucidate mechanisms involved in pituitary tumorigenesis and progression, we performed individual gene expression microarray analysis using Affy U133 Plus 2.0 GeneChips comparing 14 gonadotrope tumors with 9 normal pituitary samples obtained at autopsy.

Dataset Information

Cytogenetic, Genomic, and Functional Characterization of Pituitary Gonadotrope Cell Lines.

Publications

Cytogenetic, Genomic, and Functional Characterization of Pituitary Gonadotrope Cell Lines.

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