Measurement of the initial levels of DNA damage in human lymphocytes induced by 29 kV X rays (mammography X rays) relative to 220 kV X rays and gamma rays

Experiments using the alkaline comet assay, which measures all single-strand breaks regardless of their origin, were performed to evaluate the biological effectiveness of photons with different energies in causing these breaks. The aim was to measure human lymphocytes directly for DNA damage and subsequent repair kinetics induced by mammography 29 kV X rays relative to 220 kV X rays, 137Cs gamma rays and 60Co gamma rays. The level of DNA damage, predominantly due to single-strand breaks, was computed as the Olive tail moment or percentage DNA in the tail for different air kerma doses (0.5, 0.75, 1, 1.5, 2 and 3 Gy). Fifty cells were analyzed per slide with a semiautomatic imaging system. Data from five independent experiments were transformed to natural logarithms and fitted using a multiple linear regression analysis. Irradiations with the different photon energies were performed simultaneously for each experiment to minimize interexperimental variation. Blood from only one male and one female was used. The interexperimental variation and the influence of donor gender were negligible. In addition, repair kinetics and residual DNA damage after exposure to a dose of 3 Gy were evaluated in three independent experiments for different repair times (10, 20, 30 and 60 min). Data for the fraction of remaining damage were fitted to the simple function F(d) = A/(t + A), where F(d) is the fraction of remaining damage, t is the time allowed for repair, and A (the only fit parameter) is the repair half-time. It was found that the comet assay data did not indicate any difference in the initial radiation damage produced by 29 kV X rays relative to the reference radiation types, 220 kV X rays and the gamma rays of 137Cs and 60Co, either for the total dose range or in the low-dose range. These results are, with some restrictions, consistent with physical examinations and predictions concerning, for example, the assessment of the possible difference in effectiveness in causing strand breaks between mammography X rays and conventional (150-250 kV) X rays, indicating that differences in biological effects must arise through downstream processing of the damage.     

Production of delayed death and neoplastic transformation in CGL1 cells by radiation-induced bystander effects

Other investigators have demonstrated by transfer of medium from irradiated cells and by irradiation with low-fluence alpha particles or microbeams that cells do not have to be directly exposed to ionizing radiation to be detrimentally affected, i.e. bystander effects. In this study, we demonstrate by transfer of medium from X-irradiated human CGL1 hybrid cells that the killing of bystander cells reduces the plating efficiency of the nonirradiated CGL1 cells by 33 +/- 6%. In addition, we show that the amount of cell death induced by bystander effects is not dependent on X-ray dose, and that the induction of apoptosis does not appear to be responsible for the cell death. Furthermore, we found that the reduction in plating efficiency in bystander cells is evident for over 18 days, or 22 cell population doublings, after medium transfer, despite repeated refeeding of the cell cultures. Finally, we report the novel observation that bystander effects induced by the transfer of medium from irradiated cells can induce neoplastic transformation. Exposing unirradiated CGL1 cells to medium from cells irradiated with 5 or 7 Gy increased the frequency of neoplastic transformation significantly from 6.3 x 10(-6) in unirradiated controls to 2.3 x 10(-5) (a factor of nearly four). We conclude that the bystander effect induces persistent, long-term, transmissible changes in the progeny of CGL1 cells that result in delayed death and neoplastic transformation. The data suggest that neoplastic transformation in bystander cells may play a significant role in radiation-induced neoplastic transformation at lower doses of X rays.     

A radiation-induced acute apoptosis involving TP53 and BAX precedes the delayed apoptosis and neoplastic transformation of CGL1 human hybrid cells

Exposing CGL1 (HeLa x fibroblast) hybrid cells to 7 Gy of X rays results in the onset of a delayed apoptosis in the progeny of the cells 10 to 12 cell divisions postirradiation that correlates with the emergence of neoplastically transformed foci. The delayed apoptosis begins around day 8 postirradiation and lasts for 11 days. We now demonstrate that the delayed apoptosis is also characterized by the appearance of approximately 50-kb apoptotic DNA fragments and caspase 3 activation postirradiation. In addition, we confirm that stabilization of TP53 and transactivation of pro-apoptosis BAX also occurs during the delayed apoptosis and show that anti-apoptosis BCL-X(L) is down-regulated. To test whether the delayed apoptosis was due to a nonfunctional acute TP53 damage response in CGL1 cells, studies of acute apoptosis were completed. After irradiation, CGL1 cells underwent an acute wave of apoptosis that involves TP53 stabilization, transactivation of BAX gene expression, and a rapid caspase activation that ends by 96 h postirradiation. In addition, the acute onset of apoptosis correlates with transactivation of a standard wild-type TP53-responsive reporter (pG13-CAT) in CGL1 cells after radiation exposure. We propose that the onset of the delayed apoptosis is not the result of a nonfunctional acute TP53 damage response pathway but rather is a consequence of X-ray-induced genomic instability arising in the distant progeny of the irradiated cells.     

Relative biological effectiveness of low-energy X-rays (25 kV) in mutant p53 cancer cells

Low-energy X-rays as used in radiation therapy and diagnostics such as mammography are associated with a certain risk of promoting tumour development, especially in patients with mutations in cancer-related genes like TP53. The present study therefore addressed the relative biological effectiveness (RBE) of low-energy X-rays for two human adenocarcinoma cell lines of the breast (MDA-MB-468) and pancreas (BxPC-3) with a mutation in the TP53 gene. Clonogenic survival and cytogenetic changes in terms of micronuclei (MN) formation were determined following irradiation with 25 kV X-rays and 200 kV reference irradiation in the dose range of 1–8 Gy. Except the frequency of MN-containing binucleated cells (BNC) (BNC?+?MN/BNC) in breast cancer cells yielding an RBE between 0.6 and 0.8, both cell lines displayed dose-dependent variations of RBE values between 1 and 2 for all biological end points (cell survival, (BNC?+?MN/BNC), MN/BNC, MN/(BNC?+?MN)) with increased effectiveness of 25 kV irradiation in pancreatic compared to breast cancer cells. The results confirm previous findings indicating increased effectiveness of low-energy X-rays and underline the necessity of careful risk estimation for cancer screening programmes.

     

Feline sarcoma virus induced in vitro progression from premalignant to neoplastic transformation of human diploid cells

Summary Human diploid cells morphologically transformed by feline sarcoma virus were serially propagated under selective cell culture conditions. When injected into nude mice prior to passage in soft agar (0.35%), morphologically transformed cells did not produce tumors. However, when propagated under selective cell culture conditions, transformed cells grew in soft agar and, when injected subcutaneously into the subcapsular region of the nμ/nμ mice, produced neoplastic nodules histopathologically interpreted as fibromas. Karyological examination of cell populations grown out from the tumors confirmed that the tumors were composed of human cells. Examination of electron micrographs of the excised tumor tissue revealed the presence of budding virus particles. Tumor cells isolated from nude mice and morphologicaly transformed cells both contained the feline concornativirus-associated cell membrane antigen. It was concluded that expression of feline oncornavirus-associated cell membrane antigen is associated with an early stage of feline rerovirus-induced carcinogenesis, namely focus formation. In addition, it was shown that FeLV-FeSV can induce morphological transformation in human cells in vitro and that there is a requirement for the cells to passage through soft agar before subsequent tumor formation (neoplastic transformation) can be demonstrated. This work was supported in part by NIH-NCI RO1-259007, NO1-CP-3571 and CPV08 103563, and Air Force F49620-77-C-110.     

Detection of translocation rob(1;29) in bull sperm using a specific DNA probe

The Robertsonian translocation rob(1;29), connected with reduced fertility, is widespread in different cattle breeds all over the world. After laser microdissection, DOP-PCR, cloning and sequencing, a highly sensitive translocation-specific DNA probe, suitable for detection of rob(1;29) in cattle metaphase and interphase cells, including spermatozoa was designed. Sperm samples of five heterozygous translocation carriers were analyzed using this probe and a control probe for chromosome 6. One thousand decondensed spermatozoa from each bull were scored. Signals of the translocation-specific probe were detected in 48.8, 50.9, 50.1, 51.8, and 54.8% of spermatozoa, respectively. In contrast, semen samples from five chromosomally normal bulls showed only signals of the control probe for chromosome 6. Semen from a chimeric (XX/XY) bull, showing 57.5% of 59,XX,rob(1;29) and 42.5% of 60,XY cells in cultured peripheral lymphocytes, was also examined using this probe. No sperm head with signal of the translocation-specific probe was observed among 1,000 spermatozoa analyzed in this bull, demonstrating that female cells do not pass through the process of spermatogenesis.     

Relation of the slow growth phenotype to neoplastic transformation: Possible significance for human cancer

Summary Deletions are widely distributed over the genome in the most frequently occurring human cancers and are the most abundant genetic lesion found there. Deletions are highly correlated with the slow growth phenotype of mutated animal and human cells and result in chromosomal transposition when the retained ends are joined. Transpositions are only a minor source of mutation in rapidly multiplying bacteria but are a major cause of mutations in stationary bacteria. The NIH 3T3 line of mouse cells undergoes neoplastic transformation during prolonged incubation in a stationary state and expresses the slow growth phenotype on serial subculture at low density, suggesting a relation between transformation and chromosomal deletions. To further explore the relation between neoplastic transformation and the slow growth phenotype as a surrogate for deletions, two sublines of the NIH 3T3 cells with differing competence for transformation were serially subcultured in the stationary state at confluence and tested at each subculture for transformation and growth rate. Cell death in a fraction of the population and a heritable slowdown in proliferation of most of the survivors became increasingly pronounced with successive rounds of confluence. The reduction in growth rate was not proportional to the degree of transformation of the cultures, but all of the transformed cultures were slow growers at low density. All of the discrete colonies from cloning transformed cultures developed at a lower initial rate than control colonies under optimal conditions for growth, but they continued to grow at later stages, forming multilayered colonies under conditions that inhibited the further growth of the control colonies. The results suggest that prolonged incubation of NIH 3T3 cells in the stationary state results in growth-impairing deletions over a wide range of sites in the genome, but more restricted subsets of such lesions are responsible for neoplastic transformation. These findings provide dynamic, functional support in culture for the histopathological evidence that the quiescent state of cells associated with atrophy and fibrosis plays a significant role in the origin of some cancers in experimental animals and human beings.     

Activation of Notch-1 signaling maintains the neoplastic phenotype in human Ras-transformed cells

Truncated Notch receptors have transforming activity in vitro and in vivo. However, the role of wild-type Notch signaling in neoplastic transformation remains unclear. Ras signaling is deregulated in a large fraction of human malignancies and is a major target for the development of novel cancer treatments. We show that oncogenic Ras activates Notch signaling and that wild-type Notch-1 is necessary to maintain the neoplastic phenotype in Ras-transformed human cells in vitro and in vivo. Oncogenic Ras increases levels and activity of the intracellular form of wild-type Notch-1, and upregulates Notch ligand Delta-1 and also presenilin-1, a protein involved in Notch processing, through a p38-mediated pathway. These observations place Notch signaling among key downstream effectors of oncogenic Ras and suggest that it might be a novel therapeutic target.     

Characterization of Wiskott–Aldrich syndrome (WAS) mutants using Saccharomyces cerevisiae

Wiskott–Aldrich syndrome (WAS) is caused by alterations in the WAS protein (WASP), and 80% of the missense mutations are located in the WH1 domain, the region essential for interaction with the WASP-interacting protein (WIP). It has been suggested that loss of WASP–WIP interaction is causal to the disease. Las17p (yeast WASP) is essential for growth at 37 °C. The growth defect of the las17 Δ strain can be suppressed by the expression of human WASP together with WIP. Using the las17 Δ strain, we have analyzed 52 missense mutations in the gene encoding WASP and found that 13 of these mutant proteins were unable to suppress the growth defect of the las17 Δ strain. The majority of these 13 mutations cause the classic WAS in humans and are located within the WH1 domain, while none of the 12 mutations outside the WH1 domain abolished the activity of WASP in Saccharomyces cerevisiae cells. This suggests that some of the mutations (13 out of 40) in the WH1 domain cause the syndrome in humans by perturbing the WASP–WIP complex formation, while the rest of the mutations cause the syndrome without affecting the WASP–WIP complex formation, but may affect the activity of the complex.     

The human T-lymphotropic virus type I tax gene can cooperate with the ras oncogene to induce neoplastic transformation of cells.

Epidemiologic studies have linked infection by the human T-lymphotropic virus type I (HTLV-I) with the development of adult T-cell leukemia. The low penetrance of the virus and the long latency for disease manifestation are factors that obscure the role of HTLV-I infection in oncogenesis. We have used an in vitro transformation assay system to determine directly whether the HTLV-I tax gene has transformation potential. Transfection of the tax gene alone into early-passage rat embryo fibroblasts did not induce morphological alterations. However, cotransfection of tax with the selectable marker plasmid pRSVneo gave rise to G418-resistant colonies that could be established as immortalized cell lines. Cotransfection of tax with the ras oncogene into rat embryo fibroblasts gave rise to foci of transformed cells that were highly tumorigenic in nude mice. These data represent a direct demonstration of the oncogenic potential of the tax gene in nonlymphoid cells and establish HTLV-I as a transforming virus.     

Effect of negative pions,relative to 140 kV - 29 MeV photons and 20 MeV electrons,on proliferation of Ehrlich ascites carcinoma cells

Summary The effect of negative pions (peak and plateau), photons (140 kV and 29 MeV), and 20 MeV electrons on the proliferative capacity of Ehrlich ascites carcinoma cells was investigated. Proliferative survival curves plotted for the modalities employed are presented. Under the experimental conditions used, the peak pions were more effective than plateau pions by a factor of about 1.4. For 50% survival, 140 kV X-rays had the same effect as peak pions but the latter was more effective (factor 1.2) at 10% survival level. When 140 kV X-rays were taken as the standard, following are the RBE values calculated at 50% survival level: plateau pions -0.73; peak pions -about 1.0; 29 MeV photons -0.73 and 20 MeV electrons -0.6. The results obtained are compared with those reported on other tumor systems and biological test objects.     

Site-specific PEGylation for high-yield preparation of Lys(21)-amine PEGylated growth hormone-releasing factor (GRF) (1-29) using a GRF(1-29) derivative FMOC-protected at Tyr(1) and Lys(12)

PEGylation has been viewed as an effective means of overcoming the therapeutic restriction of growth hormone-releasing factor (1-29) (GRF(1-29)) due to its short biological lifetime caused by severe proteolysis and rapid glomerular filtration. Of three isomers according to the PEGylation sites (Tyr1, Lys12, or Lys21), PEGylated GRF(1-29) at Lys21-amine (Lys21-PEG-GRF(1-29)) was shown to have the highest bioactivity. In this report, we propose a unique two-step site-specific PEGylation method capable of producing only Lys21-PEG-GRF(1-29) with a single composition in high yield using a GRF(1-29) derivative protected at Tyr1 and Lys12 and remained available at Lys21 (FMOC1,12-GRF(1-29)). The first step of this reaction involved the PEG attachment to FMOC1,12-GRF(1-29), and the second step involved the removal of FMOC moieties. This PEGylation process was optimized at the following conditions: 0.2-0.3% (v/v) triethylamine concentration, 5.0-6.0-fold molar amount of PEG, reaction temperature of 25-45 degrees C, and reaction time of 30 min. Under these conditions, the maximum yield of Lys21-PEG-GRF(1-29) produced was ca. approximately 95%, 6.3-fold higher than that by nonspecific PEGylation at pH 8.5. Significantly, this site-specific Lys21-PEG-GRF(1-29) was found to have greatly increased resistance to rat plasma, liver, and kidney homogenates, with 7.0-, 25.4-, and 16.4-fold longer half-lives vs GRF(1-29), respectively. Furthermore, 125I-Lys21-PEG-GRF(1-29) displayed significantly reduced liver and kidney distributions and extended blood presence vs 125I-GRF(1-29) in rats. Due to these benefits, Lys21-PEG-GRF(1-29) displayed an enhanced initial growth hormone release in vivo despite having 15% remaining activity in vitro. This devised PEGylation method using an FMOC-protection/deprotection strategy would provide great usefulness for PEGylating bioactive peptides in terms of improved biological potency, elevated production yield, and a uniform composition.     

Small intestinal differentiation in human colon carcinoma HT29 cells has distinct effects on the lateral diffusion of lipids (ganglioside GM1) and proteins (HLA class 1, HLA class 2, and neoplastic epithelial antigens) in the apical cell membrane

We have studied the effect of maturation to small intestinal-like epithelial cells of the human colonic carcinoma cell line HT29 on the lateral mobility of different representative membrane components (lipid, proteins), as assessed with fluorescence recovery after photobleaching (FRAP). Maturation was induced in vitro in the HT29 cells by replacing glucose (Glu) with galactose (Gal) in the growth medium (DMEM) during a 21-day period. Scanning electron microscopy revealed an increased number of microvilli in the apical cell membrane, and enzyme analyses (alkaline phosphatase, aminopeptidase) in combination with aqueous countercurrent distribution, indicated that maturation was induced with DMEM-Gal. In comparison to control cells grown in DMEM-Glu medium, the more small intestinal-like cells grown in DMEM-Gal displayed no alteration of the lateral mobility of either cholera toxin (B subunit)-labelled ganglioside GM1 (diffusion coefficient, D [x 10(8)] = 0.8-0.9 cm2s-1; mobile fraction, R = 50-60%) or antibody-stained Class 2 histocompatibility (HLA-DR) antigen (D [x 10(9)] = 2 cm2s-1; R = 60-70%). However, antibody-labelled beta 2-microglobulin of HLA Class 1 antigen displayed increased mobility in HT29-Gal cells; D was x 1.4 and R x 1.8 larger in the HT29-Gal cells. By contrast, the mobility of a neoplastic antigen was reduced; D and R were x0.60 and x0.69 of the values seen in HT29-Glu cells. It is thus concluded that DMEM-Gal-induced differentiation in confluent HT29 cells is accompanied by specific rather than general effects on the lateral mobility of different membrane components.     

Sensitivity of a human hybrid cell line (HeLa X skin fibroblast) to radiation-induced neoplastic transformation in G2, M, and mid-G1 phases of the cell cycle

The dependence of gamma-radiation-induced neoplastic transformation frequency on position in the cell cycle was measured for a human hybrid cell line (HeLa X skin fibroblast). The end point used was the induction of a tumor-associated antigen which in these cells correlates with tumorigenicity. Induction was measured in cells at G2, M, and mid-G1 phases and compared with the frequency induced in asynchronous cells. For studies of cells in G2 phase, the cells of an asynchronous population were collected for 3 h post-irradiation using the mitotic shake-off technique. For studies of cells in M and mid-G1 phases, cells were collected by mitotic harvest and then treated at the appropriate time. The data show that cells in G2 and M phase are very radiosensitive in terms of both cell killing and induction of neoplastic transformation compared to cells in mid-G1 or asynchronous populations. At a dose of 1 Gy, the transformation frequency was 10- to 20-fold higher for cells in M and G2 phase than for cells in mid-G1 or for asynchronous cells. However, the data indicate that the transformation frequencies were similar in the different phases of the cell cycle when correlated with surviving fraction. The results indicate that transformation frequency is more sensitive to changes in dose than is cell survival.     

Re-evaluation of the endocranial volume of the Guattari 1 Neandertal specimen (Monte Circeo)

The endocranial capacity of Guattari 1 originally was estimated by Sergi as approximately 1.550 cm3. Using three different approaches, a physical endocast, a stereolithographic model, and a virtual endocast, we have estimated the endocranial capacity of Guattari 1 as approximately 1.350 cm3. This paper explains our revision of the estimated endocranial volume of Guattari 1, provides a cautionary case concerning other estimates of endocranial volume, and demonstrates and encourages the use of recent advances in imaging, modeling, and analysis of endocranial volume.