While conducting a search for cell cycleregulated 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 G 2 cell cycle checkpoint. Overexpression of HSIX1 in MCF7 cells abrogated the G 2 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 tumorigenesis͞tumor progression, possibly through a cell cycle function.Homeodomain-containing proteins act as transcription factors that regulate the coordinated expression of genes involved in both development and differentiation. They were identified initially in Drosophila, where they were found to be important in the control of segment identity (1). The genes encoding homeodomain proteins (homeobox genes) contain a common 183-nt sequence encoding a 61-aa domain that is responsible for DNA binding (2). They are postulated to act as a network of transcriptional regulators effecting cell-cell communication during normal development, alterations of which may contribute to the neoplastic phenotype. Recent studies have demonstrated homeobox gene involvement in leukemias (3) and in solid tumors such as breast, kidney, lung, and colon (4).In this study, we cloned the HSIX1 homeobox gene from late S phase mammary carcinoma cells and demonstrated that overexpression of HSIX1 leads to an abrogation of the DNA damage-induced G 2 cell cycle checkpoint. In addition, overexpression of HSIX1 occurs in a large percentage of mammary carcinomas and correlates strongly with metastatic breast disease. Preliminary tests on several cancer cell lines suggest that HSIX1 may be overexpressed in multiple types of tumors. This study links HSIX1 to the cell cycle as well as to tumor progression and lends further credence to the hypothesis that ''master regulators'' involved in development may contribute to tumorigenicity. MATERIALS AND METHODSCell Culture and Synchronization. 21PT, NT, MT1, and MT2 breast cancer cells were derived from a patient with an infiltrating and intraductal carcinoma (5) and were obtained from the laboratory of Ruth Sager (Dana-Farber Cancer Institute). The cells were cultured at 37°C in 6.5% CO 2 in ␣-MEM plus 10% fetal bovine serum͞2 mM L-glutamine͞1 mM sodium pyruvate͞0.1 mM nonessential amino acids͞10 mM Hepes͞1 g/ml insulin͞12.5 ng/ml epidermal growth factor͞1 g/ml hydrocortisone, and antibiotics. The generated MCF7 transfectants were cultured in RPMI 1640 medium plus 10% FBS͞600 g/ml G418, and antibiotics. Normal luminal and myoepithelial cells were sorted by immunomagnetic methods fro...

We show here that mammalian site-specific recombination and DNA-repair pathways share a common factor. The effects of DNA-damaging agents on cell lines derived from mice homozygous for the scid (severe combined immune deficiency) mutation were studied. Surprisingly, all scid cell lines exhibited a profound hypersensitivity to DNAdamaging agents that caused double-strand breaks (xirradiation and bleomycin) but not to other chemicals that caused single-strand breaks or cross-links. Neutral filter elution assays demonstrated that the x-irradiation hypersensitivity could be correlated with a deficiency in repairing double-strand breaks. These data suggest that the scid gene product is involved in two pathways: DNA repair of random doublestrand breaks and the site-specific and lymphoid-restricted variable-(diversity)-joining [V(D)J] DNA rearrangement process. We propose that the scid gene product performs a similar function in both pathways and may be a ubiquitous protein.Mice homozygous for the scid (severe combined immune deficiency) mutation lack a functional immune system but otherwise appear normal (1). The absence of B and T lymphocytes in scid mice is due to a defect in the site-specific V(D)J recombination pathway that is responsible for the somatic assembly of immunoglobulin and T-cell receptor genes. Analysis of scid variable (diversity) joining [V(D)J] recombination events has shown that large deletions, which remove all or most of the coding sequences of immunoglobulin or T-cell receptor genes, accompany the rearrangements and result in nonfunctional lymphoid cells (2-4). Furthermore, examination of model rearrangement templates (recombinant retroviruses and plasmids) introduced into scid lymphoid cells has recapitulated the aberrant deletional rearrangements (4-6). Thus, we and others have proposed that the scid gene product is an integral component of the V(D)J recombinase complex.Mutations that affect site-specific and general recombination frequently also affect the pathways responsible for repairing DNA double-strand breaks (DSBs) due to chromosome damage (7). This overlap of recombination and DSBrepair pathways is presumed to result from the postulated role of double-stranded ends as structural intermediates in many types of recombination and repair (for review, see ref.8). DSBs can also be generated by a number of DNAdamaging agents, the most common of which is ionizing radiation. X-ray-induced DSBs can stimulate chromosomal deletions and aberrant rearrangements and are lethal if not repaired (9). We show here that the similarity between recombination and DSB-repair pathways extends to mammalian cells affected by the scid mutation. scid cells were found to be hypersensitive specifically to agents that make DSBs. In addition, a dynamic assay for DNA repair demonstrated that the scid mutation severely diminished DSB repair. We propose that the scid gene product performs a similar function in both the V(D)J recombination and DSBrepair pathways. MATERIALS AND METHODSFibroblastic Cell Lines. Fib...

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