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CytogeneticsCytogenetics

Cytogenetics

Figure 1: Fluorescence in situ hybridization analysis of NCEB-1 mantle cell lymphoma

Cytogenetics informs both authentication and characterization of cancer cell lines. Because authentication principally rests on DNA-STR profiling, for most cell lines, notably older classics, their defining identifier is uniqueness. Cytogenetic data provide independent verification of DNA profiling by anchoring present with published karyotypes, which for cancer cell lines are also effectively unique

Thanks to mapping data and clone resources provided in the wake of the Human Genome Project, molecular cytogenetics also informs oncogenomic characterization via identification of target genes at recurrent chromosome translocation breakpoints.

The methods used at the DSMZ to address the all-too-often complex chromosome rearrangements present in cancer cells include classical karyotyping followed by fluorescence in situ hybridization (FISH) summarized in Ref1.

The Blind Men and the Elephant

Cells in culture present a rather homogeneous picture which must be clarified and enlarged by further study. Cell lines keep their secrets well however, and are still capable of surprises. The NCEB-1 mantle cell lymphoma cell line (2) pictured here was established back in the ‘eighties (Figure 1). Yet it took another 20 years before its true hybrid nature was disclosed by cytogenetic analysis (3). NCEB-1, whose cytogenetic composition is basically human (red centromeres), also carries 6-8 mouse chromosomes (green). The presence of mouse chromosomes in NCEB-1 stocks from different labs points to a cell fusion event early in the life of the cell line, probably in the laboratory of its originators who used a mouse macrophage feeder layer at early passage. The significance of the seemingly stable mouse component remains a matter for speculation (4).

Without cytogenetic analysis, it is unlikely that the hybrid nature of NECB-1 would have come to light, implying a need for multiple techniques, such as gene mutational and expression profiling, to see the whole picture. Like those blind men each holding different parts of an elephant , only a joint picture makes sense in the end.

Cell Lines Have Got Real (at Last)

In cancer cell labs intraspecies cell line cross-contamination (CLCC) is now a recognized problem. Several thousand different human cell lines are now in circulation, any of which might contaminate any other, raising both the risk and the difficulty of identifying of CLCC.

Figure 2: FISH analysis of BCL11B rearrangement in a t-cell leukemia cell line HPB-ALL

In 1999, using cytogenetics supported by DNA fingerprinting, about one-in-six “new” cell lines were shown to becross-contaminated (5). Thanks, however, to the coordinated efforts of cell banks the risk of encountering CLCC among new cell lines has now fallen dramatically. Alas, the risk of CLCC among “second hand” cell lines remains unacceptably high.

Cell lines are unique resources for cancer gene discovery and investigating which pathways are thereby deregulated (6). An example is provided by BCL11B, which was first characterized in cell lines established from T-cell leukemia where it juxtapositionally activates NK-family homeobox genes (TLX3 and NKX2-5) in recurrent chromosome translocations. Figure 2 shows rearrangement of BCL11B in a T-cell leukemia cell line (7).

NB: While closely following the guidelines for cytogenetic nomenclature set out in ISCN (8, 9) a number of minor modifications to these conventions are forced by the nature and extent of rearrangements sometimes encountered among continuous cell lines. For clarity and ease of data manipulation, numerical changes are described separately from, and precede, structural changes. Parentheses following modal chromosome numbers surround upper and lower ranges of chromosome counts (ignoring broken or heteroploid cells). Some frequently used abbreviations are explained as follows: abr, abnormally banded region; cen, centromere; colon single (:), break (in detailed descriptions); colon double (::), breakage and reunion (in detailed description); add, additional material of unknown origin; del, deletion; der, derivative chromosome; dic, dicentric; dir, direct; dmin, double minute; dup, duplication; hsr, homogeneously staining region; ins, insertion; inv, inversion; mar, marker chromosome; min, minute; minus (-), loss of; p, short arm; Ph, Philadelphia chromosome; plus (+), gain of; psu, pseudo; q, long arm; question mark (?), questionable chromosome identification; r, ring chromosome; rcp, reciprocal; s, satellite; semicolon (;), separates chromosomes/regions in structural rearrangements involving more than one chromosome; t, translocation; ter, terminal.

References:
1. MacLeod RAF, Kaufmann M, Drexler HG: Cytogenetic harvesting of commonly used tumor cell lines. Nature Protocols 2: 372-382 (2007).
2. Saltman DL, Cachia PG, Dewar AE, Ross FM, Krajewski AS, Ludlam C, et al.: Characterization of a new non-Hodgkin’s lymphoma cell line (NCEB-1) with a chromosomal (11:14) translocation [t(11:14)(q13;q32)]. Blood 72: 2026–2030 (1988).
3. Camps J, Salaverria I, Garcia MJ, Prat E, Be`a S, Pole JC, et al.: Genomic imbalances and patterns of karyotypic variability in mantle cell lymphoma cell lines. Leukemia Res 30: 923–934 (2006).
4. Drexler HG and MacLeod RAF. Mantle cell lymphoma-derived cell lines: Unique research tools. Leukemia Res 30: 911-913 (2006).
5. MacLeod RAF, Dirks WG, Kaufmann M, Matsuo Y, Milch H, Drexler HG: Widespread intraspecies cross-contamination of human tumor cell lines arising at source. Int J Cancer 83: 555-563 (1999).
6. MacLeod RAF, Nagel S, Scherr M, Schneider B, Dirks WG, Uphoff CC, Quentmeier H, Drexler HG: Human leukemia and lymphoma cell lines as models and resources. Current Medicinal Chemistry 15: 339-359 (2008).
7. MacLeod RAF, Nagel S, Kaufmann M, Janssen JWG, Drexler HG: Activation of HOX11L2 by juxtaposition with 3'-BCL11B in an acute lymphoblastic leukemia cell line (HPB-ALL) with t(5;14)(q35;q32.2). Genes Chromosomes Cancer 37: 84-91 (2003).
8. Harnden DG, Klinger HP: An international system for human cytogenetic nomenclature. Cytogenetics and Cell Genetics. Basel (1985).
9. Mitelman F (ed): ISCN: Guidelines for Cancer Cytogenetics. Supplement to an International System for Human Cytogenetic Nomenclature. Basel, S. Karger (1991).