title: T24 HRAS transformed NIH/3T3 mouse cells (GhrasT-NIH/3T3) in serial tumorigenic in vitro/in vivo passages give rise to increasingly aggressive tumorigenic cell lines T1-A and T2-A and metastatic cell lines T3-HA and T4-PA.
abstract: Cancer cells often arise progressively from "normal" to "pre-cancer" to "transformed" to "local metastasis" to "metastatic disease" to "aggressive metastatic disease". Recent whole genome sequencing (WGS) and spectral karyotyping (SKY) of cancer cells and tumorigenic models have shown this progression involves three major types of genome rearrangements: ordered small step-wise changes, more dramatic "punctuated evolution" (chromoplexy), and large catastrophic steps (chromothripsis) which all occur in random combinations to generate near infinite numbers of stochastically rearranged metastatic cancer cell genomes. This paper describes a series of mouse cell lines developed sequentially to mimic this type of progression. This starts with the new GhrasT-NIH/Swiss cell line that was produced from the NIH/3T3 cell line that had been transformed by transfection with HRAS oncogene DNA from the T24 human bladder carcinoma. These GhrasT-NIH/Swiss cells were injected s.c. into NIH/Swiss mice to produce primary tumors from which one was used to establish the T1-A cell line. T1-A cells injected i.v. into the tail vein of a NIH/Swiss mouse produced a local metastatic tumor near the base of the tail from which the T2-A cell line was established. T2-A cells injected i.v. into the tail vein of a nude NIH/Swiss mouse produced metastases in the liver and one lung from which the T3-HA (H=hepatic) and T3-PA (P=pulmonary) cell lines were developed, respectively. T3-HA cells injected i.v. into a nude mouse produced a metastasis in the lung from which the T4-PA cell line was established. PCR analysis indicated the human T24 HRAS oncogene was carried along with each in vitro/in vivo transfer step and found in the T2-A and T4-PA cell lines. Light photomicrographs indicate that all transformed cells are morphologically similar. GhrasT-NIH/Swiss cells injected s.c. produced tumors in 4% of NIH/Swiss mice in 6-10 weeks; T1-A cells injected s.c. produced tumors in 100% of NIH/Swiss mice in 7-10 days. T1-A, T-2A, T3-HA and T4-PA cells when injected i.v. into the tail produced local metastasis in non-nude or nude NIH/Swiss mice. T4-PA cells were more widely metastatic than T3-HA cells when injected i.v. into nude mice. Evaluation of the injected mice indicated a general increase in metastatic potential of each cell line in the progression as compared to the GhrasT-NIH/3T3 transformed cells. A new photomicrographic technique to follow growth rates within six preselected 2×2mm(2) grids per plate is described. Average doubling times of the transformed cells GhrasT-NIH/3T3 (17h), T1A (17.5h), T2A (15.5h), T3-HA (17.5h) and T4-PA (18.5h) (average 17.2h) were significantly faster (P=0.006) than NIH Swiss primary embryonic cells and NIH/3T3 cells (22 h each). This cell series is currently used in this lab for studies of cancer cell inhibitors, mitochondrial biogenesis and gene expression and is available for further study by other investigators for intra- and inter-laboratory comparisons of WGS, transcriptome sequencing, SKY and other analyses. The genome rearrangements in these cells together with their phenotypic properties may help provide more insights into how one tumorigenic progression occurred to produce the various cell lines that led to the highly metastatic T4-PA cell line.
authors.affiliation: Department of Biology, Grove City College, 100 Campus Drive, Grove City, PA 16127, USA; Department of Biochemistry, Oral Roberts University School of Medicine, 7777 S Lewis Ave, Tulsa, OK 74171, USA. Electronic address: raydb@gcc.edu.
authors.forename: Durwood B
authors.initials: DB
authors.lastname: Ray
authors.affiliation: Department of Biology, Grove City College, 100 Campus Drive, Grove City, PA 16127, USA. Electronic address: Merrilljer@aol.com.
authors.forename: Gerald A
authors.initials: GA
authors.lastname: Merrill
authors.affiliation: Department of Biology, Grove City College, 100 Campus Drive, Grove City, PA 16127, USA. Electronic address: BrennerFJ@gcc.edu.
authors.forename: Frederic J
authors.initials: FJ
authors.lastname: Brenner
authors.affiliation: Department of Biology, Grove City College, 100 Campus Drive, Grove City, PA 16127, USA. Electronic address: LSLytle@gcc.edu.
authors.forename: Laurie S
authors.initials: LS
authors.lastname: Lytle
authors.affiliation: Department of Biochemistry, Oral Roberts University School of Medicine, 7777 S Lewis Ave, Tulsa, OK 74171, USA. Electronic address: tanmlam@aol.com.
authors.forename: Tan
authors.initials: T
authors.lastname: Lam
authors.affiliation: Department of Biology, Grove City College, 100 Campus Drive, Grove City, PA 16127, USA. Electronic address: aaron.mcelhinney@msn.com.
authors.forename: Aaron
authors.initials: A
authors.lastname: McElhinney
authors.affiliation: Department of Biology, Grove City College, 100 Campus Drive, Grove City, PA 16127, USA. Electronic address: jba6776@yahoo.com.
authors.forename: Joel
authors.initials: J
authors.lastname: Anders
authors.affiliation: Department of Biology, Grove City College, 100 Campus Drive, Grove City, PA 16127, USA. Electronic address: taramtauber@yahoo.com.
authors.forename: Tara Tauber
authors.initials: TT
authors.lastname: Rock
authors.affiliation: Department of Biology, Grove City College, 100 Campus Drive, Grove City, PA 16127, USA. Electronic address: jenlyker@gmail.com.
authors.forename: Jennifer Kier
authors.initials: JK
authors.lastname: Lyker
authors.affiliation: Department of Biology, Grove City College, 100 Campus Drive, Grove City, PA 16127, USA. Electronic address: scott.barcus@gmail.com.
authors.forename: Scott
authors.initials: S
authors.lastname: Barcus
authors.affiliation: Department of Biology, Grove City College, 100 Campus Drive, Grove City, PA 16127, USA. Electronic address: karaleslie31@gmail.com.
authors.forename: Kara Hust
authors.initials: KH
authors.lastname: Leslie
authors.affiliation: Department of Biology, Grove City College, 100 Campus Drive, Grove City, PA 16127, USA. Electronic address: jkramer@buffalo.edu.
authors.forename: Jill M
authors.initials: JM
authors.lastname: Kramer
authors.affiliation: Department of Biology, Grove City College, 100 Campus Drive, Grove City, PA 16127, USA. Electronic address: emrubenstein@bsu.edu.
authors.forename: Eric M
authors.initials: EM
authors.lastname: Rubenstein
authors.affiliation: Department of Biology, Grove City College, 100 Campus Drive, Grove City, PA 16127, USA. Electronic address: kkschanz@yahoo.com.
authors.forename: Karen
authors.initials: K
authors.lastname: Pryor Schanz
authors.affiliation: Department of Biology, Grove City College, 100 Campus Drive, Grove City, PA 16127, USA. Electronic address: amyjparkhurst@gmail.com.
authors.forename: Amy J
authors.initials: AJ
authors.lastname: Parkhurst
authors.affiliation: Department of Biology, Grove City College, 100 Campus Drive, Grove City, PA 16127, USA. Electronic address: Peckmichellea@gmail.com.
authors.forename: Michelle
authors.initials: M
authors.lastname: Peck
authors.affiliation: Department of Biology, Grove City College, 100 Campus Drive, Grove City, PA 16127, USA. Electronic address: kimberlyross@gmail.com.
authors.forename: Kimberly
authors.initials: K
authors.lastname: Good
authors.affiliation: Department of Biology, Grove City College, 100 Campus Drive, Grove City, PA 16127, USA. Electronic address: granathkl@gmail.com.
authors.forename: Kristi Lemke
authors.initials: KL
authors.lastname: Granath
authors.affiliation: Department of Biology, Grove City College, 100 Campus Drive, Grove City, PA 16127, USA. Electronic address: cifranm1@gmail.com.
authors.forename: Nicole
authors.initials: N
authors.lastname: Cifra
authors.affiliation: Department of Biology, Grove City College, 100 Campus Drive, Grove City, PA 16127, USA. Electronic address: mattnjessd@gmail.com.
authors.forename: Jessalee Wantz
authors.initials: JW
authors.lastname: Detweiler
authors.affiliation: Department of Biology, Grove City College, 100 Campus Drive, Grove City, PA 16127, USA. Electronic address: laura.stevens2011@gmail.com.
authors.forename: Laura
authors.initials: L
authors.lastname: Stevens
authors.affiliation: Department of Biology, Grove City College, 100 Campus Drive, Grove City, PA 16127, USA. Electronic address: rma165@psu.edu.
authors.forename: Richard
authors.initials: R
authors.lastname: Albertson
authors.affiliation: Department of Biology, Grove City College, 100 Campus Drive, Grove City, PA 16127, USA. Electronic address: rd41193n@pace.edu.
authors.forename: Rachael
authors.initials: R
authors.lastname: Deir
authors.affiliation: Department of Biology, Grove City College, 100 Campus Drive, Grove City, PA 16127, USA. Electronic address: elisabeth.e.stewart@gmail.com.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Stewart
authors.affiliation: Department of Biology, Grove City College, 100 Campus Drive, Grove City, PA 16127, USA. Electronic address: wingardkl1@gmail.com.
authors.forename: Katherine
authors.initials: K
authors.lastname: Wingard
authors.affiliation: Department of Biology, Grove City College, 100 Campus Drive, Grove City, PA 16127, USA. Electronic address: RichardsonMR1@gcc.edu.
authors.forename: Micah Rose
authors.initials: MR
authors.lastname: Richardson
authors.affiliation: Department of Biology, Grove City College, 100 Campus Drive, Grove City, PA 16127, USA. Electronic address: BlizardSB1@gcc.edu.
authors.forename: Sarah B
authors.initials: SB
authors.lastname: Blizard
authors.affiliation: Department of Biology, Grove City College, 100 Campus Drive, Grove City, PA 16127, USA. Electronic address: GillespieLE1@gcc.edu.
authors.forename: Lauren E
authors.initials: LE
authors.lastname: Gillespie
authors.affiliation: Department of Biology, Grove City College, 100 Campus Drive, Grove City, PA 16127, USA. Electronic address: cekrieley@gcc.edu.
authors.forename: Charles E
authors.initials: CE
authors.lastname: Kriley
authors.affiliation: Department of Biology, Grove City College, 100 Campus Drive, Grove City, PA 16127, USA. Electronic address: Rzewnickidi1@gcc.edu.
authors.forename: Daniel I
authors.initials: DI
authors.lastname: Rzewnicki
authors.affiliation: Department of Biology, Grove City College, 100 Campus Drive, Grove City, PA 16127, USA; Department of Biochemistry, Oral Roberts University School of Medicine, 7777 S Lewis Ave, Tulsa, OK 74171, USA. Electronic address: j.jeandave@gmail.com.
authors.forename: David H
authors.initials: DH
authors.lastname: Jones
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic / Q000235:genetics* / Q000473:pathology*; D018450:Disease Progression; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000473:pathology / Q000556:secondary; D008175:Lung Neoplasms / Q000235:genetics / Q000473:pathology / Q000556:secondary; D051379:Mice; D008819:Mice, Nude; D041681:NIH 3T3 Cells; D009362:Neoplasm Metastasis / Q000235:genetics* / Q000473:pathology*; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics; D001749:Urinary Bladder Neoplasms / Q000235:genetics* / Q000473:pathology*
chemical_list: C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)
keywords: Chromoplexy; Chromothripsis; Doubling time method; GhrasT-NIH/3T3; HRAS; In vitro/in vivo tumorigenic progression; Metastasis; NIH/3T3; Punctuated evolution; Tansformed