Cytogenetic assays for mitotic poisons: the diploid Chinese hamster cell system

When the diploid Chinese hamster cells (line Don) were treated with Colcemid (final concentrations 0.05 micrograms/ml and 2 mg/ml) for three hours and the mitotic arrest was reversed by washing and reincubation in the control medium, the cells were able to reorganize spindle and divide within 2-4 hr. When the cells were treated with vinblastine (concentrations 10(-6) and 10(-4) M), there was a continuous accumulation of mitoses after vinblastine was removed. At 10(-6) M, a small proportion of anaphase figures was observed in reversed cell populations but at 10(-4) M, there was no anaphase. The results suggest that tubulin was precipitated or destroyed even in cells in interphase. The G2 and S cells were able to complete their cell cycle but were unable to divide because of the lack of microtubules. When recovery was prolonged to 24, 48 and 72 hr, the cell populations exhibited higher percentages of polyploid and aneuploid elements than the control cell populations. There appeared to be a nonrandom increase of the small metacentric than the larger chromosomes. This test system, after protocol improvements, should be useful to assay mitotic poisons that are water soluble or soluble in DMSO.     

Partial trisomy of the long arm of chromosome 1 in myelofibrosis and polycythemia vera

We have identified partial trisomy 1q in 2 patients with different hematologic disorders. The first patient was a 55-year-old female with myelosclerosis and myeloid metaplasia diagnosed at age 38 years presenting with anemia, fatigue, bruising, fever, and splenomegaly. At age 56, she had 50–95% myeloblast cells and 95–100 nucleated RBC precursors per 100 WBC. Chromosome analysis of unstimulated leukocytes with Q, G, and C banding showed 46,XX,-6,+t(1;6) (q25;p22) in all metaphase cells. In vitro incorporation of Fe⁵⁵ was demonstrated in 90% of metaphases by autoradiography. The second patient, a 49-year-old male, was diagnosed as having polycythemia vera at age 30 during a regular checkup. He since developed hepatosplenomegaly. Chromosome analysis from a direct bone marrow preparation at age 44 and 45 showed grossly normal karyotypes. At age 49, his marrow by Q and G banding showed almost 100% of cells with 46,XY,–13,+t(1;13) (q12;p12). Eleven cases of trisomy of 1q have been reported in various hematologic disorders. It is apparent that partial trisomy 1q represents another nonrandom chromosomal abnormality, in addition to the most common nonrandom chromosomal aberrations, such as the Philadelphia chromosome, trisomy 8, trisomy 9, and monosomy 7 in hematologic disorders.     

Inoculation of vector cell monolayers with potato yellow dwarf virus

Results from inoculation of two vector-specialized forms of potato yellow dwarf virus (PYDV) on cell monolayers derived from their vectors and related nonvectors indicate that the effect of pH on inoculation is mediated through the virus, not through the host. The optimum pH for sanguinolenta yellow dwarf virus (SYDV) inoculation on cell monolayers is near 5.9; the optimum pH for inoculation of constricta yellow dwarf virus (CYDV) on cell monolayers is near 5.3. Forty-five minutes is sufficient for inoculation of PYDV on leafhopper cell monolayers. A pH of 7.0 is better than 6.5 or 7.5 for inoculating leaves of Nicotiana rustica with SYDV. Use of histidine-MgCl₂ (His-MgCl₂) solution for inoculation of SYDV on N. rustica gave better results than did phosphate buffer at the same pH.In His-MgCl₂ buffer SYDV is most stable near pH 6.4 at 0° and pH 6.5 at 30°; CYDV is most stable near pH 6.9 at 0° and pH 5.9 at 30°. Near the optimal pH for inoculation of cell monolayers with SYDV at 30°, namely pH 5.9, the half-life of the virus in the His-MgCl₂ solution is 21.4 min. The corresponding half-life for CYDV near its optimal pH for inoculation, namely 5.3, is 7.7 min.Both SYDV and CYDV reached peak concentrations in N. rustica about 10 days after the appearance of the first systemic symptom. After reaching such a peak about 30% of the infectious SYDV was lost by day 12, but thereafter the concentration remained almost constant up to day 24. Similarly, about 70% of peak CYDV infectivity was lost by day 12, and more than 80% by day 14 and day 16.Purified sterile infectious inocula were obtained by harvesting infected N. rustica plant tissue at the time of peak virus concentration. SYDV can be stored at ?80° for as long as 3 years without appreciable loss of infectivity. CYDV, similarly prepared and stored, seriously deteriorated in infectivity between 6 and 12 months after storage.     

17beta-Estradiol upregulates and activates WOX1/WWOXv1 and WOX2/WWOXv2 in vitro: potential role in cancerous progression of breast and prostate to a premetastatic state in vivo

Human WWOX gene encodes a proapoptotic WW domain-containing oxidoreductase WOX1 (also named WWOX, FOR2 or WWOXv1). Apoptotic and stress stimuli activate WOX1 via Tyr33 phosphorylation and nuclear translocation. WOX1 possesses a tetrad NSYK motif in the C-terminal short-chain alcohol dehydrogenase/reductase (SDR) domain, which may bind estrogen and androgen. Here, we determined that 17beta-estradiol (E(2)) activated WOX1, p53 and ERK in COS7 fibroblasts, primary lung epithelial cells, and androgen receptor (AR)-negative prostate DU145 cells, but not in estrogen receptor (ER)-positive breast MCF7 cells. Androgen also activated WOX1 in the AR-negative DU145 cells. These observations suggest that sex hormone-mediated Tyr33 phosphorylation and nuclear translocation of WOX1 is independent of ER and AR. Stress stimuli increase physical binding of p53 with WOX1 in vivo. We determined here that E(2) increased the formation of p53/WOX1 complex and their nuclear translocation in COS7 cells; however, nuclear translocation of this complex could not occur in MCF7 cells. By immunohistochemistry, we determined that progression of prostate from normal to hyperplasia, cancerous and metastatic stages positively correlate with upregulation and activation of WOX1 and WOX2 (FOR1/WWOXv2). In contrast, breast cancer development to a premetastatic state is associated with upregulation and Tyr33 phosphorylation of cytosolic WOX1 and WOX2, followed by significant downregulation or absent expression during metastasis. These Tyr33-phosphorylated proteins are mostly located in the mitochondria without translocating to the nuclei, which is comparable to those findings in cultured breast cancer cells. Together, sex steroid hormone-induced activation of WOX1 and WOX2 is independent of ER and AR, and this activation positively correlates with cancerous progression of prostate and breast to a premetastatic state.     

The MCT-1 oncogene product impairs cell cycle checkpoint control and transforms human mammary epithelial cells

Multiple copies in T-cell maligancy (MCT-1) is a putative oncogene initially identified in a human T-cell lymphoma. Forced expression of MCT-1 has recently been shown to induce cell transformation and proliferation, as well as to activate survival-related PI-3K/AKT pathways protecting cells from apoptosis. MCT-1 protein is stabilized in response to DNA damage. The impact of MCT-1 overexpression on DNA damage response remains unknown. Here, we show that MCT-1 deregulates cell cycle checkpoints. The phosphorylation of genomic stabilizers H2AX and NBS1 are enhanced in MCT-1-overexpressing cells. Forced expression of MCT-1 significantly increases the number of DNA damage-induced foci involving gamma-H2AX and 53BP1. In MCT-1-overexpressing cells, the proportion of S-phase cell population is preferentially increased after exposure to gamma-irradiation compared to controls. Knockdown of endogenous MCT-1 using an siRNA approach attenuates the H2AX phosphorylation and the G1/S checkpoint defect. Furthermore, MCT-1 is capable of transforming immortalized human mammary epithelial cells and promoting genomic instability. These data shed light on the role of MCT-1 in the cellular response to DNA damage and its involvement in malignant transformation.