Catalytic subunit of DNA-dependent protein kinase: impact on lymphocyte development and tumorigenesis - PubMed (original) (raw)

Catalytic subunit of DNA-dependent protein kinase: impact on lymphocyte development and tumorigenesis

A Kurimasa et al. Proc Natl Acad Sci U S A. 1999.

Abstract

The DNA-dependent protein kinase (DNA-PK) consists of a heterodimer DNA-binding complex, Ku70 and Ku80, and a large catalytic subunit, DNA-PKcs. To examine the role of DNA-PKcs in lymphocyte development, radiation sensitivity, and tumorigenesis, we disrupted the mouse DNA-PKcs by homologous recombination. DNA-PKcs-null mice exhibit neither growth retardation nor a high frequency of T cell lymphoma development, but show severe immunodeficiency and radiation hypersensitivity. In contrast to the Ku70-/- and Ku80-/- phenotype, DNA-PKcs-null mice are blocked for V(D)J coding but not for signal-end joint formation. Furthermore, inactivation of DNA-PKcs leads to hyperplasia and dysplasia of the intestinal mucosa and production of aberrant crypt foci, suggesting a novel role of DNA-PKcs in tumor suppression.

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Figures

Figure 1

Figure 1

Inactivation of DNA-PKcs by homologous recombination. (A) Schematic diagram of the murine DNA-PKcs locus from exons 1 to 10 and hybridization probe (Top), the targeting construct (Middle), and the targeted allele. _Bam_HI (B), _Eco_RI (E), and Hin_dIII (H) restriction sites are indicated. (B) Southern blot of the_Bam_HI-digested tail DNA from control wild-type (WT), heterozygous (+/−), and homozygous (−/−)DNA-PKcs_-targeted mice. The wild-type and mutant fragments are 10 and 2.2 kb, respectively. (C) RT-PCR of 5′-(exon 1–4) and 3′-(PI-3 kinase domain) regions of_DNA-PKcs RNA from wild-type, DNA-PKcs-targeted, and SCID mouse cells. Total RNA was isolated from SV40-transformed lung fibroblast cells. PCR reactions were performed with (+) or without (−) reverse transcriptase (RT). RT-PCR for GAPDH was performed to ensure RNA integrity. (D) Western blot analysis of the various cells. Whole-cell extracts were prepared from primary and SV40-transformed lung fibroblast cells. Anti-DNA-PKcs monoclonal antibody and anti-Ku70 polyclonal antibody were used for detection. Note that there is another gene, MCM4, which is located about 700 bp upstream of DNA-PKcs (32). The transcription of DNA-PKcs and MCM4 is independently controlled by two distinct promoters located in this 700-bp region. We have carefully designed the DNA-PKcs knockout vector in exon 3, which is about 10 kb away from the promoter region, thus avoiding any possibility of interfering with the expression of the MCM4 gene. Furthermore, we have shown that aberrant DNA-PKcs mRNA is expressed in_DNA-PKcs−/− mice, confirming that the promoter region of the DNA-PKcs gene is not affected by our knockout construct.

Figure 2

Figure 2

Development of lymphocytes is blocked at early stages in DNA-PKcs−/− mice. (A) Histological analysis of thymus (Thy), spleen (Spl) and lymph node (LN) from wild-type and DNA-PKcs−/− mice (×200). Tissue sections were stained with hematoxylin and eosin. In tissue samples from DNA-PKcs_-deficient mice, we observed effacement of normal histology and replacement by immature cells. The abbreviations are as follows: C, cortex; M, medulla; W, white pulp; R, red pulp; GC, germinal center. (B) Flow cytometric analysis of cells from the thymus (Thy), bone marrow (BM), and spleen (Spl) for the presence of precursor and mature T and B cells. Thymocytes and splenocytes were stained with fluorochrome-conjugated antibodies to CD4 and CD8; splenocytes and BM cells were stained with fluorochrome-conjugated antibodies to B220 and IgM or CD43. Profiles shown are representative results from a 4- to 5-week-old DNA-PKcs−/− mouse, its heterozygous littermate, and an age-matched CB-17 SCID mouse. (C) TCR and Ig gene rearrangement in_DNA-PKcs−/− mice. (a) TCRβ rearrangement by PCR analysis. Thymus and spleen DNA were assayed for recombination of Vβ8-Jβ2.6. Both the quantity and the diversity of TCRβ rearrangement were reduced in DNA-PKcs−/− and SCID mice. (b) Coding joint of TCRδ rearrangement. Thymus and spleen DNA were assayed for recombination of Dδ2-Jδ1. (c) Signal joint of TCRδ rearrangement. Thymus DNA was assayed for Dδ2-Jδ1 circular signal joint products. There is more amplified signal for both DNA-PKcs−/− and SCID mice than for heterozygous control mice. (e) Ig heavy-chain rearrangement by PCR analysis. BM and spleen DNA were used for recombination of VH7183-JH4. Rearrangement in DNA-PKcs−/− and SCID is severely reduced in both BM and spleen. (d) and (f) Control GAPDH amplification from thymus, spleen, and BM DNA. DNA (100, 10, or 1 ng) from thymus, spleen, and BM or from a 5-week-old_DNA-PKcs+/−_ mouse (lanes 1–3), of a 9-week-old_DNA-PKcs+/−_ mouse (lane 4–6), and 100 ng DNA of three individual DNA-PKcs−/− mice (lanes 7–9) and three individual SCID mice (lanes 10–12).DNA-PKcs−/− and SCID mice analyzed were also between 4 and 9 weeks of age.

Figure 3

Figure 3

Radiation-dose response of_DNA-PKcs−/−_ cells. Clonogenic survival was measured on SV40-transformed mouse lung fibroblasts irradiated with graded doses of ionizing radiation. DNA-PKcs−/− cells show similar sensitivity to ionizing radiation as SCID and are much more sensitive than wild-type (+/+) and heterozygous (+/−) cells.

Figure 4

Figure 4

Preneoplastic lesions in_DNA-PKcs−/−_ mice. Intestinal tissue samples from 6-week- to 6-month-old DNA-PKcs−/− mice were sectioned, stained with hematoxylin and eosin, and photographed. (A) Section of intestinal tissue showing inflammation and mild epithelial hyperplasia (×100). (B) Photomicrograph of colonic mucosa showing crypt hyperplasia with mild to moderate dysplasia (×200). (C) Adenomatous polyp of the colon showing areas of severe dysplasia (×400). (D) Aberrant crypt foci along the intestinal mucosa showing severe dysplasia (×400). (E) Section of intestinal tissue from a wild-type mouse showing normal morphology (×250).

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