Mutation of Pten/Mmac1 in mice causes neoplasia in multiple organ systems

Abstract

Pten/Mmac1+/- heterozygous mice exhibited neoplasms in multiple organs including the endometrium, liver, prostate, gastrointestinal tract, thyroid, and thymus. Loss of the wild-type allele was detected in neoplasms of the thymus and liver. Surprisingly, tumors of the gastrointestinal epithelium developed in association with gut lymphoid tissue. Tumors of the endometrium, thyroid, prostate, and liver were not associated with lymphoid tissue and appeared to be highly mitotic. In addition, these mice have nonneoplastic hyperplasia of lymph nodes that was caused by an inherited defect in apoptosis detected in B cells and macrophages. Examination of peripheral lymphoid tissue including lymphoid aggregates associated with polyps revealed that the normal organization of B and T cells was disrupted in heterozygous animals. Taken together, these data suggest that PTEN is a regulator of apoptosis and proliferation that behaves as a "landscaper" tumor suppressor in the gut and a "gatekeeper" tumor suppressor in other organs.

Figure 1

Targeted disruption of Pten in mice. (a) Restriction map of Pten and the targeting construct. Restriction sites are as follows: KpnI (K), BglII (Bg), SacI (ScI), EcoRI (RI). The targeting construct contains a cassette of the phosphoglycerate kinase promoter upstream of the neomycin resistance gene ORF (PGK-NEO), which has replaced the BglII fragment containing the phosphatase domain in exon 5. (b) Southern blot of mouse tail DNA digested with SacI and hybridized with probe B. The samples loaded are the offspring of a chimera × C57BL6 mating. The two lanes on the left have only the wild-type band while the two lanes on the right have both the wild-type and mutant bands. (c) RT-PCR analysis of Pten. RNA was prepared from W9.5 embryonic stem cells and the livers of +/+ and +/− littermates. RNAs were either reverse transcribed (+) or placed in mock reactions (−), and the entire ORF was amplified with a single pair of primers. After electrophoresis on a 1% agarose gel and staining with ethidium bromide, the expected wild-type 1.2-kb band was detected in all samples subjected to reverse transcription. The sample from the +/− mouse also produced a product just under 1 kb.

Figure 2

Immunohistochemical protein expression patterns of PTEN in developing mouse embryos: day 8 (a, b), day 9 (c), day 10 (d), day 11 (e), day 13 (f), day 14 (g), and day 16 (h). In embryos from day 8 through approximately day 10, PTEN expression was minimal in tissues such as yolk sac (YS), neural tube (NT), somite (S), amnion (A), body wall (BW), optic vesicle (OV), and embryonic gut (G), yet was routinely positive in placenta (P) (a–d). From day 11, embryos expressed PTEN widely at moderate levels, including in lung (L), muscle (M), cartilage (C), vertebral body (VB), rib cartilage primordium (R), and marginal layer of the spinal cord (SC-M); enhanced expression was observed in heart (H), dorsal mantle layer of the spinal cord (SC-Mt), and epidermis (Ep). i was a day 11 embryo section stained with preimmune serum. Erythrocytes (E) displayed little or no staining at any stage. Other structures were brain ventricle (V) and dorsal aorta (DA). Scale bar = 1 mm.

Figure 3

Histological survey of tissues in Pten+/− mice. (a) Papillary thyroid carcinoma of 18-week-old Pten+/− male, hematoxylin/eosin (×400). (b) Anti-Ki67 stain of the same thyroid carcinoma. (c) A malignant lymphoma of the kidney of six-month-old Pten+/− male (case 87), hematoxylin/eosin (×200). (d and e) Immunohistochemical analysis of proliferation using anti-Ki67 antibody on prostates of +/+ (d) and +/− (e) mice. Arrow points to mitotic figure (×200). (f) Prostatic intraepithelial neoplasia found in 17-week-old Pten+/− male, hematoxylin/eosin (×400). (g–i) Mouse uteri. (g) Cross-section of the uterus of the 26-week-old wild-type mouse. Arrow points to the endometrium. Arrowhead points to the normal gland (×40). (h) Cross-section of the uterus of the 31-week-old heterozygous mouse. Arrow points to the endometrium. Arrowhead points to the atypical hyperplastic gland (×40). (i) Arrowhead points to a region of complex atypical hyperplasia in a 29-week-old heterozygous mouse (×100). (j) Decreased survival of the Pten heterozygous animals. (k) Loss of heterozygosity of Pten in tumors. Southern blots of normal-tumor pairs of three independent T-cell lymphomas analyzed with probe B. DNA was digested with SacI.

Figure 4

Gross and histological appearance of gut polyps and lymphoid hyperplasia. (a and b) The gastrointestinal tract was cut longitudinally along the antimesenteric border and cleaned. Polyps were visualized by eye and then inspected with a dissecting microscope. Contrast was enhanced with india ink. (a) A 5-mm polyp (×6) at the colorectal boundary. (b) Two polyps in the jejunum of diameters of 2.5 and <1 mm (×20). (c–e) Clean mouse intestines were rolled and embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Arrow indicates luminal side of the lymphoid aggregate. (c) Lymphoid aggregate in the colon of the wild-type mouse (×100). (d) Lymphoid aggregate in the small intestine of the wild type (×40). (e) Lymphoid aggregate in the small intestine of the heterozygous mouse (×100). Note association of polyp growth with the Peyer’s patch. (f) A pedunculated polyp of the colon with hyperplasia and a lymphoid aggregate in the center (×40). (g) Hyperplastic polyp of the jejunum (×100). (h) Retention polyp of the colon (×40). (i) Pten+/− female with lymphadenopathy of neck and axilla (arrows). (j and k) Paraffin sections of (j) paracortical and medullary effacement and (k) paracortical effacement and follicular hyperplasia of the heterozygous mice (×40).

Figure 5

Analysis of lymphoid abnormalities in Pten heterozygous mice. (a) Penetrance of the lymph node hyperplasia in different age and sex groups of Pten+/− mice (82 females, 86 males studied). (b and c) Flow cytometric analysis of cells from four lymphoid organs. Cells from three wild-type and three heterozygous animals labeled with various surface markers as indicated on the horizontal axis and then labeled with either annexin V antibody (b) or Ki67 antibody (c). One asterisk indicates P < 0.05, two asterisks indicate P < 0.01. (d–g) Immunohistochemical analysis of lymph nodes and lymphoid aggregates of the gut of the wild-type and heterozygous animals. CD3-positive cells appear brown and B220-positive cells appear blue. Arrows point to the lumen side of the intestinal wall. Arrowheads point to the germinal centers. (d and e) Lymph node of the wild-type (d) and heterozygous (e) mouse. (f and g) Colonic lymphoid aggregates of the wild-type (f) and heterozygous (g) mice.