Analysis of gene alterations of mitochondrial DNA D-loop regions to determine breast cancer clonality.

BACKGROUND: It has been a challenge to determine breast cancer clonality accurately. The aim of the present study was to assess methods using formalin-fixed paraffin-embedded (FFPE) tissue to differentiate new primary tumours from true recurrences that are associated with poorer prognoses and often require more aggressive treatment. METHODS: We investigated the novel method of analysing gene alterations of mitochondrial DNA D-loop region (GAMDDL) and compared it with the conventional method of analysing the X-chromosome-linked human androgen receptor (HUMARA). The FFPE sections of primary and secondary breast cancers, the non-neoplastic mammary gland, and lymph nodes were examined. RESULTS: Informative rates for HUMARA, GAMDDL, and combined analyses were 42.1%, 76.9%, and 89.5%, respectively. All of the 10 contralateral breast cancers were determined to be non-clonal. In contrast, 3 out of 8 (37.5%) of the ipsilateral secondary tumours shared a clonal origin with the primary tumour and were classified as true recurrences, whereas 4 out of 8 (50%) were classified as new primary tumours. CONCLUSION: GAMDDL analysis represents a novel and useful molecular method for examining the precise cell lineages of primary and secondary tumours, and was more accurate than HUMARA in determining clonality.

Expression of interleukin-15 and its receptor by human fetal retinal pigment epithelial cells.

PURPOSE: IL-15 and IL-15 receptor expression was measured in retinal pigment epithelial (RPE) cells to support a possible role of IL-15 in ocular inflammatory and immune responses. METHODS: Reverse transcription-coupled polymerase chain reaction (RT-PCR) and Northern blot analysis of IL-15 mRNA in previously characterized non-transformed and simian virus (SV)-40 transformed human fetal RPE cells were carried out. Biological activities of IL-15 produced by the RPE cells were assayed by co-culture with IL-15 responsive cells. Expression of the IL-15 receptor (IL-15R) alpha, IL-2R beta and gamma chains were examined by RT-PCR. RESULTS: Both non-transformed and SV-40 transformed human fetal RPE cells express IL-15, a T cell growth factor which has similar biological activities to IL-2, and the expression of IL-15 is enhanced by interferon-gamma (IFN-gamma) or tumor necrosis factor-alpha (TNF-alpha) stimulation. In addition, transcripts for all three IL-15 receptor components (IL-15R alpha, IL-2R beta and IL-2R gamma) were detected in these cells. CONCLUSIONS: RPE cells produce IL-15, which may play an important role in ocular immune and inflammatory responses by stimulating infiltrated T cells and RPE cells via paracrine and autocrine loops, respectively.

Reconstitution of a functional interleukin (IL)-7 receptor demonstrates that the IL-2 receptor gamma chain is required for IL-7 signal transduction.

The interleukin (IL)-2 receptor gamma chain has recently been shown to be a component of the IL-7 and IL-4 receptors. Using a transient transfection assay and the trans-activation of reporter gene constructs which are under the control of cytokine-responsive promoter elements, we have studied signal transduction through the IL-7 receptor (IL-7R). The reporter gene expression was not stimulated by receptors that contained the cytoplasmic domain of the IL-7R, either as intact IL-7R or as part of a chimeric receptor. However, co-expression of the IL-7R with the IL-2 receptor gamma chain was able to stimulate gene activation. For maximal stimulation the intact cytoplasmic domains of each chain was required.

The action of interleukin-2 receptor subunits defines a new type of signaling mechanism for hematopoietin receptors in hepatic cells and fibroblasts.

The gene regulatory functions of the human IL-2 receptor (IL-2R) were reconstituted in transiently transfected hepatoma cells. The combination of IL-2R beta and -gamma mediated a strong stimulation via the cytokine response element of the alpha 1-acid glycoprotein gene and the hematopoietin receptor response element, but none via the IL-6 response element or the sis-inducible element. IL-2R alpha enhanced 10-fold the sensitivity of the IL-2R beta.gamma complex to respond to IL-2 or IL-15, but did not modify the specificity or the magnitude of maximal gene regulation. A homodimerizing chimeric receptor G-CSFR-IL-2R beta could mimic the IL-2R action. The IL-2R-mediated gene regulation was similar to that seen with receptors for IL-4 and IL-7, but differed from that for IL-6 type cytokines, thrombopoietin, erythropoietin, and growth hormone. The activation of STAT proteins by the IL-2R was assessed in transfected L-cells and COS-1 cells. Although IL-2R subunits were highly expressed in these cells, no STAT protein activation was detectable. Transient overexpression of JAK3 was unable to change the signaling specificity of the hematopoietin receptors in rat hepatoma, L-, and COS cells, but established a prominent activation of the IL-6 response elements by the IL-2R and IL-4R in HepG2 cells. The data support the model that the IL-2R and related hematopoietin receptors produce at least two separate signals which control gene expression.