Luteinizing hormone receptor deficiency increases the susceptibility to alkylating agent-induced lymphomagenesis in mice

Abstract

Previous studies have revealed a close link between luteinizing hormone (LH)/human chorionic gonadotropin (hCG) signaling and oncogenesis in gonadal and nongonadal tissues. To investigate whether genetic ablation of LH receptor (Lhr) affects the animal's oncogenic susceptibility, adult female wild-type (wt), heterozygous, and homozygous Lhr knockout (LhrKO) mice were intraperitoneally injected with an alkylating agent, N-methyl-N-nitrosourea (MNU, 50 mg/kg of body weight). The mice were sacrificed when they were short of breath or 10 months after the injection. The results showed that MNU induced non-Hodgkin's thymic and lymphonodus lymphomas in 70.6% and 100% of heterozygous and homozygous animals, respectively, compared with 35.7% in wt siblings. The tumor development was rapid; they were more aggressive and metastasized to the spleen, liver, and kidney in Lhr-deficient mice compared to wt siblings. All tumors were immunostained-positive for a T-cell specific marker, CD3, but not for a B-cell marker, CD22, suggesting that all the lymphomas arose from T-cells, which are known to be LH/hCG receptor-positive. There was no rearrangement of the Lhr gene locus or differences in thymic cell proliferation among the genotypes. However, apoptosis was lower in the Lhr-deficient thymuses. The thymic Bcl-2 levels were elevated and caspase-3 activation was reduced in Lhr heterozygous and homozygous animals. In conclusion, MNU induced a higher incidence and an earlier onset of aggressive lymphomas in LhrKO animals, which may be associated with a reduction in apoptosis of thymocytes.

Keywords: Apoptosis; Gene knockout; LH/hCG receptor; Lymphoma; MNU; Thymus.

Fig. 1

MNU-induced lymphoma in LhrKO female mice. a The gross appearance of enlarged thymus (yellow arrow) and cervical lymphoids (white arrow) in an LhrKO homozygous female mouse sacrificed 3 months after a single dose of MNU injection. b The percent of tumor-free animals after MNU injection

Fig. 2

MNU induces non-Hodgkin’s lymphomas. Lymphomas in the thymus (a–c), lymph node (d–f), spleen (g–i), liver (j–l), and kidney (m–o) are all stained positive for a T-cell marker, CD3, but negative for a B-cell marker, CD22. Magnification: a and f, ×300; b–e and g–o, ×150

Fig. 3

Southern blotting performed on EcoRI digested genomic DNA isolated from normal and thymus tumors showed that wild-type and mutant alleles in the tumor tissues were the same as in the normal tissues

Fig. 4

Immunohistochemical staining for a cell proliferation marker, PCNA, in normal thymuses (a–c) showed no genotype differences among the genotypes (a–c). TUNEL assay in normal thymuses (d–f) revealed a decrease in clusters of apoptotic cells in heterozygous (+/−) and homozygous (−/−) animals compared with wild-type siblings (+/+). Magnification: a–f, ×150; insets in a–c, ×600

Fig. 5

Semiquantitative RT–PCR and Western blotting revealed that mRNA (a) and protein (b) levels of Bcl-2, an anti-apoptotic factor, in both normal thymuses and thymic tumors of heterozygous (+/−) and homozygous (−/−) mice were significantly higher than in wild-type (+/+) siblings. n = 6; ^a p < 0.001, compared with normal thymuses of +/− and −/− animals and tumors of all genotypes; ^b p < 0.05, compared with normal thymuses of wild-type animals

Fig. 6

Western blotting demonstrated that inactive caspase-3 levels are not significantly different among the genotypes in either normal thymus or thymic tumor. The active caspase-3, on the other hand, markedly decreased in the normal thymuses of heterozygous and homozygous mice compared to wild-type siblings. Although active caspase-3 levels in thymic tumors were not different among the genotypes, they were all significantly lower than in normal thymuses of wild-type animals. n = 6; ^a p < 0.05, ^b p < 0.01, compared with normal thymuses of wild-type siblings