Abrogation of the G2 cell cycle checkpoint associated with overexpression of HSIX1: a possible mechanism of breast carcinogenesis

Abstract

While conducting a search for cell cycle-regulated genes in human mammary carcinoma cells, we identified HSIX1, a recently discovered member of a new homeobox gene subfamily. HSIX1 expression was absent at the onset of and increased toward the end of S phase. Since its expression pattern is suggestive of a role after S phase, we investigated the effect of HSIX1 in the G2 cell cycle checkpoint. Overexpression of HSIX1 in MCF7 cells abrogated the G2 cell cycle checkpoint in response to x-ray irradiation. HSIX1 expression was absent or very low in normal mammary tissue, but was high in 44% of primary breast cancers and 90% of metastatic lesions. In addition, HSIX1 was expressed in a variety of cancer cell lines, suggesting an important function in multiple tumor types. These data support the role for homeobox genes in tumorigenesis/tumor progression, possibly through a cell cycle function.

Figure 1

Differential expression of HSIX1 throughout S phase of 21PT cells. (A) Pulsed ³H-thymidine incorporation after release from mimosine arrest shows progression of cells through S phase. (B) Section of the differential display gel demonstrating the differential expression of 6A (subsequently identified as HSIX1) in S phase. (C) Northern blot analysis confirming the differential expression of HSIX1 throughout S phase of 21PT cells. RNA was isolated from cells after release from mimosine arrest, and Northern blot analysis was performed with the HSIX1 cDNA probe. (Lower) Ethidium bromide (EtBr) staining as a loading control. The numbers 2, 4, 6, and 8 represent time in hours after release from mimosine block.

Figure 2

Overexpression of HSIX1 in MCF7 cells abrogates the G₂ cell cycle checkpoint. (A) Northern blot analysis of HSIX1 transfectants and controls. Clones labeled HSIXA were stably transfected with the SIXFL plasmid; those labeled CATB were stably transfected with the parent vector containing the CAT gene [pcDNA3.1(CAT)]. Note that HSIXA2, while G418-resistant, does not express HSIX1. (Lower) EtBr staining as a loading control. (B) Representative FACS analysis on propidium iodide-stained cells before and after x-ray irradiation at a dose of 8 Gy. The panels display a time course after irradiation of one HSIX1 transfectant (HSIXA13) and one control transfectant (CATB3) cell line. The experiment was performed several times at two different doses of irradiation (5 and 8 Gy) with the same outcome. (C) Summary of the percentage of cells in G₂ at various time points before and after irradiation in the transfectants and controls. The data graphed are from one experiment performed at 8 Gy and are representative of several experiments performed at 5 and 8 Gy. Note that cells expressing HSIX1 progress through the G₂ arrest at a more rapid rate than transfected controls.

Figure 3

HSIX1 is not expressed in normal breast, but is expressed in normal skeletal muscle, salivary gland, lung, trachea, and kidney. A Human RNA Master Blot from CLONTECH was probed with the HSIX1 cDNA. (A) HSIX1 expression pattern in normal tissues. (B) Identities of all the samples included on the blot. (C) The blot was stripped and reprobed with ubiquitin to ensure equal loading.

Figure 4

Expression of HSIX1 correlates with metastatic disease. (A) Northern blot analysis of normal mammary epithelial cells (70N) and the 21T series of mammary carcinoma cells. 21PT and 21NT cells were derived from a primary tumor, whereas 21MT1 and 21MT2 were established from a pleural effusion from the same patient (7). (B) Quantitative representation of HSIX1 expression in the cell lines after normalization to actin. (C) Quantitative representation of a Northern blot analysis of 3 control tissues (normal adjacent breast, normal luminal cells, and normal myoepithelial cells; lanes 1, 2, and 3, respectively), 25 primary breast tumor biopsy samples (lanes 4–28), and 10 metastatic lesions (lanes 29–38). The blot was stripped and reprobed with 36B4 (16) for normalization, and relative HSIX1 expression was plotted. A 3-fold increase over normal adjacent breast was considered positive for HSIX1 and is marked by a dashed line. Forty-four percent of primary and 90% of metastatic lesions express greater than a 3-fold increase in HSIX1 mRNA over the normal adjacent breast control. It is not known whether the patients that expressed HSIX1 in the primary tumor went on to develop metastatic disease.

Figure 5

HSIX1 is expressed in other cancers. Northern blot analysis of multiple cancer cell lines. A human cancer cell line Northern blot containing 2 μg of poly(A)⁺ RNA from each cell line was probed with HSIX1 cDNA. HL-60, promyelocytic leukemia; HeLa S3, cervical cancer; K-562, chronic myelogenous leukemia; MOLT-4, lymphoblastic leukemia; Raji, Burkitt’s lymphoma; SW480, colorectal carcinoma; A549, lung carcinoma; G361, melanoma. Actin mRNA is shown as a loading control.