Epigenetic variation at the SLC6A4 gene promoter in mother-child pairs with major depressive disorder.

BACKGROUND: Genetic and epigenetic variations of the serotonin transporter gene (SLC6A4) have been related to the etiology of depression. The 5-HTTLPR polymorphism at the SLC6A4 promoter region has two variants, a short allele (S) and a long allele (L), in which the S allele results in lower gene transcription and has been associated with depression. The short S-allele of 5-HTTLPR polymorphism of this gene has been associated with depression. In addition to molecular mechanisms, exposure to early life risk factors such as maternal depression seems to affect the development of depression in postnatal life. The present study investigated the association of 5-HTTLPR polymorphism and CpG DNA methylation (5mC) levels of an AluJb repeat element at the SLC6A4 promoter region in mother-child pairs exposed to maternal depression. METHODS: We analyzed DNA samples from 60 subjects (30 mother-child pairs) split into three groups, with and without major depression disorder (DSM-IV) among children and mothers. The genotyping of 5-HTTLPR polymorphism and quantification of 5mC levels was performed by qualitative PCR and methylation-sensitive restriction enzyme digestion, and real-time quantitative PCR (MSRED-qPCR), respectively. RESULTS: The sample analyzed presented a higher frequency of S allele of 5-HTTLPR (67.5%). Despite the high frequency of this allele, we did not find statistically significant differences between individuals carrying at least one S allele between the depression and healthy control subjects, or among the mother-child pair groups with different patterns of occurrence of depression. In the group where the mother and child were both diagnosed with depression, we found a statistically significant decrease of the 5mC level at the SLC6A4 promoter region. LIMITATIONS: The limitations are the relatively small sample size and lack of gene expression data available for comparison with methylation data. CONCLUSION: In this study, we demonstrated a repeat element specific 5mC level reduction in mother-child pairs, concordant for the diagnosis of depression.

Comparing vegetation types and anthropic disturbance levels in the Atlantic forest: how do Pentatomoidea (Hemiptera: Heteroptera) assemblages respond?

The Atlantic Forest (AF) is considered the most fragmented and endangered Brazilian biome. The diversity of phytophagous insects increases after disturbances in forests, and it was hypothesized the Pentatomidae can furnish ecologically reliable information in terms of diversity in response to the changes occurring in AF. Our aim was to quantify the response of assemblages of Pentatomoidea to gradient of human disturbance in two vegetation types of the AF-dense ombrophilous forest (DOF) and mixed ombrophilous forest (MOF). Twelve transects were grouped into environmental classes, namely open, intermediate, and closed. Overall, 1,017 pentatomoids were sampled, representing 64 species. The open environment was more abundant than closed environment, though it is expected that Pentatomoidea respond with increasing abundance when under light or moderate disturbance. The MOF was more abundant than DOF, and the composition differed between both of them. Given the differences in composition between MOF and DOF, abiotic variables are important factors acting as environmental filters for Pentatomoidea, not just directly on the insects, but probably also on the nutritional support of their host plants.

Dosimetry of very high energy electrons (VHEE) for radiotherapy applications: using radiochromic film measurements and Monte Carlo simulations.

Very high energy electrons (VHEE) in the range from 100-250 MeV have the potential of becoming an alternative modality in radiotherapy because of their improved dosimetry properties compared with MV photons from contemporary medical linear accelerators. Due to the need for accurate dosimetry of small field size VHEE beams we have performed dose measurements using EBT2 Gafchromic® film. Calibration of the film has been carried out for beams of two different energy ranges: 20 MeV and 165 MeV from conventional radio frequency linear accelerators. In addition, EBT2 film has been used for dose measurements with 135 MeV electron beams produced by a laser-plasma wakefield accelerator. The dose response measurements and percentage depth dose profiles have been compared with calculations carried out using the general-purpose FLUKA Monte Carlo (MC) radiation transport code. The impact of induced radioactivity on film response for VHEEs has been evaluated using the MC simulations. A neutron yield of the order of 10(-5) neutrons cm(-2) per incident electron has been estimated and induced activity due to radionuclide production is found to have a negligible effect on total dose deposition and film response. Neutron and proton contribution to the equivalent doses are negligible for VHEE. The study demonstrates that EBT2 Gafchromic film is a reliable dosimeter that can be used for dosimetry of VHEE. The results indicate an energy-independent response of the dosimeter for 20 MeV and 165 MeV electron beams and has been found to be suitable for dosimetry of VHEE.

Efeito analgésico e anti-inflamatório do extrato aquoso das folhas de trevo-roxo (Scutellaria agrestis A. St. -Hil. ex Benth. - Lamiaceae) em roedores / Analgesic and anti-inflammatory effect of the aqueous extract of "trevo-roxo" (Scutellaria agrestis A. St. -Hil. ex Benth. - Lamiaceae) leaves in rodents

Scutellaria agrestis é utilizada por comunidades ribeirinhas do Amazonas principalmente para o tratamento de otites por via tópica utilizando-se o extrato bruto obtido por maceração. O presente trabalho visou investigar preliminarmente o perfil fitoquímico, a segurança toxicológica e as ações analgésica, anti-inflamatória e antiedematogência do extrato aquoso das folhas de S. agrestis. Foram coletados 80 indivíduos da espécie no horto medicinal da Universidade Nilton Lins, Manaus, Brasil. O perfil fitoquímico foi obtido por meio de prospecção da droga vegetal para heterosídeos cianogênicos, terpenos, compostos fenólicos e alcaloides. A toxicologia foi avaliada pelo teste de toxicidade aguda. As atividades analgésicas/ anti-inflamatórias foram analisadas por meio dos testes de formalina em camundongos e a atividade antiedematogência, pelo teste de edema de pata em ratos. Os metabólitos detectados foram fenóis (taninos hidrolisáveis, cumarinas e várias classes de flavonoides) e terpenos (esteroides livres, saponinas). Não foi possível estabelecer DL50, haja visto que o extrato não provocou a morte de nenhum animal durante o teste de toxicidade aguda, provavelmente devido à ausência de heterosídeos cianogênicos na sua composição. Apesar de não provocar morte, considerou-se que o extrato apresenta uma discreta toxicidade, uma vez que foi observada a ocorrência de espasmos na primeira hora de observação dos animais. O extrato apresentou ainda efeito analgésico e anti-inflamatório significativo nas doses de 30, 100 e 300 mg/kg pelo teste da formalina, sendo o resultado na maior dose equivalente ao obtido com a droga padrão (fentanil). No entanto, não observamos efeito antiedematogênico nas doses testadas durante as 5 horas de registro do edema de pata. Os resultados obtidos nesta pesquisa conferem base científica preliminar quanto à segurança e ao efeito analgésico e antiinflamatório da droga vegetal, o que indica que tal espécie é promissora e expressamente recomendada para maiores estudos farmacológicos in vitro e in vivo.

The Scutellaria agrestis is used by Amazonas riverine communities, especially for otitis externa topical treatment, by using the crude extract obtained by maceration. This study aimed to investigate the preliminary phytochemical profile, the safety/toxicity and the analgesic, anti-inflammatory and antiedematogenic activities of the aqueous extract of the S. agrestis leaves. Eighty individuals were collected at the Nilton Lins University medicinal garden, Manaus, Brazil. The phytochemical profile was obtained through a plant drug survey for cyanogenic heterosides, terpenes, alkaloids and phenolic compounds. The extract safety was evaluated by acute toxicity test. Analgesic and anti-inflammatory activities were accessed using formalin test in mice and the antiedematogenic activity, using paw edema test in mice. We detected phenolic (hydrolysable tannins, coumarins and several classes of flavonoids) and terpenoid (free steroids, saponins) metabolites. We could not establish LD50 because no animals died during the acute toxicity test, probably because of the absence of cyanogenic glycosides on the composition of the extract. However, we found that the extract is slightly toxic as animal spasms were observed in the first hour of the test. The extract showed significant analgesic and anti-inflammatory activity on the formalin test (30, 100 and 300 mg/kg p.o.), and the highest dose result was equivalent to the standard drug (Fentanyl). However, no significant antiedematogenic effect was observed during the paw edema test. The results obtained in this study provide preliminary scientific basis about the safety and analgesic/anti-inflammatory actions of the aqueous extract of S. agrestis, which indicates that this species is a promising option for further in vitro and in vivo pharmacological studies.

Morphoanatomy of the flower of Syagrus inajai (SPRUCE) Becc. (Arecaceae- Arecoideae- Attaleinae), Amazon.

The occurrence of Syagrus inajai (Spruce) Becc., popularly known as pupunha palm, among other names, has been registered in the Guianas and in the North of Brazil in areas of terra firme (non-flooding) and gallery forests. In order to characterize the inflorescence and further knowledge of this family, a morphoanatomical study was carried out of the palm S. inajai in a green area of the Campus of the Federal University of Amazonas--UFAM, Manaus, Amazonas. The inflorescences are branched to one order, pedunculate, and interfoliar, measuring 62-82 cm in length, with woody bracts with longitudinal grooves on the external surface, and flowers in triads. The number of flowers to each inflorescence varies from 5,904 to 17,316 for staminate flowers, and from 180 to 3,528 for pistillate flowers. Staminate flowers with six anthers and one vascular bundle each; three-lobed pistillodium, vascularized pistillodium. Its pistillate flowers have six staminodia joined to form a circle, syncarpic, tricarpellary, trilocular gynoecium, one ovule to each locule, synascidiate in the ovary, and plicated above. Tripartite stigma, apical and sessile, with epidermis composed of elongated papillary cells, pattern of epidermis that is maintained throughout the stylar canal. Bitegmented, anatrope, pachychalazal ovule.

Aspectos Estruturais e Fitoquímicos de partes vegetativas de Costus spicatus (Jacq. ) Sw. (Costaceae) / Structural and Phytochemical aspect from vegetative part of Costus spicatus (Jacq. ) Sw (Costaceae)

As folhas de Costus spicatus são amplamente empregadas na medicina popular para o tratamento de várias doenças entre elas: malária, hepatite e doença do aparelho urinário. O objetivo deste trabalho foi identificar aspectos da anatomia dos órgãos vegetativos (folhas, caules, raízes e rizomas) associados à triagem fitoquímica visando contribuir com informações relevantes para o desenvolvimento de estudos taxonômicos e farmacológicos. A análise anatômica por meio da microscopia óptica e de varredura evidenciou folha anfi-hipoestomática, com estômatos e tricomas tectores filamentosos simples. O mesofilo é constituído por parênquima clorofiliano, que se divide em duas regiões intercaladas por cordão de fibras e feixes vasculares. O caule é do tipo atactostélico como no rizoma. A raiz é poliarca. Os testes histoquímicos indicaram a presença de amido, proteínas estruturais, alcaloides, cristais de oxalato de cálcio. A prospecção química com extratos hidroalcoólico e aquoso constatou a presença de saponinas, taninos, alcaloides, compostos fenólicos e heterosídeos cianogênicos.

The leaves of Costus spicatus are widely employed in folk medicine for the treatment of several diseases, including: malaria, hepatitis and urinary tract disease. The purpose of this paper was to identify aspects of the anatomy of vegetative organs (leaves, stems, roots and rhizomes) associated with phytochemical screening to contribute with relevant information for the development of taxonomic and pharmacological studies. The anatomic analysis through optical microscopy and scanning showed amphistomatic leaves with tetracitic type stomats and simple filamentous tector trichomes. Mesophyll is constituted by a chlorophyllian parenchyma, which is divided into two regions intersected by a strand of fibers and vascular bundles. The stem is atactostelic, such as for the rhizome. The root is polyarc. The histochemical tests indicated the presence of structural proteins, alkaloids, and calcium oxalate crystals. Chemical prospecting with hydroalcoholic and aqueous extracts attested the presence of saponins, tannins, alkaloids, phenolic compounds and heterosides as cyanogenic glucosides.

Comportamento alimentar de cabras alimentadas com diferentes teores de fibra em detergente neutro / Feeding behavior of goats fed with different NDF levels

Avaliou-se o comportamento alimentar de cabras alimentadas com dietas que continham diferentes teores de fibra em detergente neutro (FDN) - 49, 54, 59, 64 e 69 por cento -, em função de diferentes teores de inclusão de resíduo úmido de cervejaria (RUC), em substituição ao concentrado da dieta - 0, 25, 50, 75 e 100 por cento. Foram utilizadas cinco cabras mestiças Boer x Saanen e cinco cabras Saanen distribuídas em dois quadrados latinos 5x5. O volumoso utilizado foi feno de tifton, com relação volumoso: concentrado de 40:60. As observações foram feitas em períodos de 20 minutos até o tempo total de 24 horas. Os animais dos diferentes genótipos não apresentaram diferenças no comportamento alimentar. Os tempos gastos com alimentação e com ociosidade não diferiram quanto aos teores de FDN na dieta, no entanto resposta linear crescente foi observada para os tempos despendidos com ruminação e com a mastigação total com o aumento dos teores de FDN e RUC na dieta. As dietas com os teores de 49 e 64 por cento apresentaram menor tempo de ruminação quando comparadas com a dieta com teor de 69 por cento de FDN, com resultados respectivos de 218, 268 e 366 minutos de ruminação. Isso contribuiu para diminuir a eficiência (min/kg MS e FDN) de alimentação, ruminação e mastigação. Conclui-se que o resíduo úmido de cervejaria pode ser usado como fonte de fibra efetiva na dieta de cabras.

The feeding behavior was evaluated in lactating goats fed diets with different levels of NDF (neutral detergent fiber) 49, 54, 59, 64, and 69 percent, in function of different levels of WBG (wet brewery grains) 0, 25, 50, 75, and 100 percent. Five goats Boer x Saanen and five Saanen goats were randomly distributed in two 5x5 latin squares. The used roughage tifton hay in a roughage:concentrate rate of 60:40. The feeding times, idle time, and rumination time were evaluated each 20 minutes during 24 hours. The feeding behavior was not influenced by genotypes. The feeding times and idles times were not affected by increasing NDF levels, however, the rumination time and total chewing time had growing linear effect with increase of NDF levels in which the levels 49 and 64 percent showed shorter rumination time than 69 percent NDF, 218, 268, and 366min respectively. It decreased the feeding, rumination and chewing efficiency (min/kg DM and NDF). It was concluded that WBG can be used as a source of effective fiber in diets of goats.

Pulmonary hilar stereotactic body radiation therapy in the rat.

Stereotactic Body Radiation therapy (SBRT) is an emerging modality of treatment for early stage non-small cell lung carcinoma. Concerns have arisen related to increased toxicities for medial tumors. We have developed a model of high dose, hypofractionated radiotherapy to the pulmonary hilum using the Leksell Gamma-Knife. Sprague-Dawley rats received hypofractionated SBRT to the unilateral lung hilum using a custom immobilization device on the Gamma Knife. Each animal was individually scanned, treatment planned, and treated with either two 4 mm or one 8 mm collimated shots at escalating doses of 20, 40, and 80 Gy to the 50% isodose volume, encompassing the right mainstem bronchus. All animals were carefully followed post-treatment and imaged by plain film and CT. In addition, histopathological analysis of all rats was performed at selected time points. Animals treated with 4 mm collimated shots demonstrated no appreciable changes on plain films or sequential, follow-up CT scans, or histopathologically. Animals irradiated with the 8 mm collimator were less active, gained weight at a reduced rate, and demonstrated histopathological changes in 7/34 animals six months post-irradiation. Cellular atypia and interstitial pneumonitis were found, three of the seven of the animals showed clear bronchial damage and two showed vascular damage. Significant volume and time effects were found. Utilizing a novel Gamma Knife based animal model to study SBRT toxicity, it was found that the bronchus will tolerate small volumes of very high dose radiotherapy. It was postulated that radiation of the surrounding support stroma and normal tissue are important in the etiology of bronchial or hilar 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.

Loss of chromosome 14 increases the radiosensitivity of CGL1 human hybrid cells but lowers their susceptibility to radiation-induced neoplastic transformation.

Loss of active tumor suppressor alleles on fibroblast chromosomes 11 and 14 are involved in radiation-induced neoplastic transformation of human hybrid CGL1 cells. Loss of either chromosome 11 or 14 alone is not sufficient for neoplastic transformation. To gain insight into the potential functions of these tumor suppressor loci, we have investigated the effects of chromosome 11 or 14 loss on radiation-induced neoplastic transformation. We recently demonstrated that loss of chromosome 11 increases the susceptibility to X-ray induced cell killing, neoplastic transformation and the expression of delayed death. The data suggested that one possible function of the chromosome 11 tumor suppressor gene may be to help maintain genome stability after radiation damage. We postulated that if the chromosome 14 allele is functioning in a similar manner, then the loss of chromosome 14 may also make the hybrid cells more susceptible to radiation-induced cell killing and neoplastic transformation. A hybrid cell line which has lost one copy of chromosome 14 was isolated and designated CON3(-14). CON3(-14) cells were more sensitive to X-ray-induced cell killing when compared with parental CGL1 cells. However, the susceptibility to radiation-induced neoplastic transformation was significantly reduced (by a factor of two) compared with the parental CGL1 cells. The expression of delayed death in the progeny of the irradiated CON3(-14) cells, growing in transformation flasks, was similar to CGL1 cells during the 21 day assay period. Taken together, the data indicate that loss of chromosome 14 alone increased the X-ray sensitivity of the hybrid cells but reduced their susceptibility to radiation-induced neoplastic transformation. These data suggest that the tumor suppressor alleles on chromosomes 11 and 14 may be functionally distinct in terms of their regulation of genomic instability and neoplastic transformation after radiation exposure.

The orphan receptor COUP-TFII regulates G2/M progression of breast cancer cells by modulating the expression/activity of p21(WAF1/CIP1), cyclin D1, and cdk2.

The orphan receptors COUP-TFI and COUP-TFII play an important role in development and differentiation by activating specific genes and by modulating the activity of nuclear receptors including estrogen receptor alpha (ERalpha) and retinoic acid receptors (RARs). Previously, it was demonstrated that the expression and activity of ERalpha and RARs are lost or impaired in anti-estrogen-resistant breast cancers. Here we show that, similar to ERalpha and RARs, the expression of COUP-TFII but not COUP-TFI is reduced in approximately 30% of breast cancer cell lines. Introduction of COUP-TFII to MDA-MB-435 cells resulted in reduced growth and plating efficiency. Interestingly, COUP-TFII increased the expression of cyclin D1 and p21(WAF1/CIP1) in MDA-MB-435 cells. Although parental and COUP-TFII-transduced cells progressed through the G1-S phase at a similar rate, progression of COUP-TFII cells through the G2/M transition phase was delayed. The activity of cdk2 required for G2/M progression was reduced in COUP-TFII cells compared to parental cells. This property of COUP-TFII is distinct from that of ERalpha and RARs, which usually modulate the G1 phase of breast cancer cells. Furthermore, these results reveal an important physiological function of COUP-TFII, which correlates with its ability to induce gene expression rather than modulation of nuclear receptor activity.

Previous loss of chromosome 11 containing a suppressor locus increases radiosensitivity, neoplastic transformation frequency and delayed death in HeLa x fibroblast human hybrid cells.

CGL1 (HeLa x fibroblast) hybrid cells have been utilized to study mechanisms of radiation-induced neoplastic transformation of human cells in vitro. Previous analysis has shown that loss of active tumor suppressor alleles on fibroblast chromosomes 11 and 14 may be required for radiation-induced neoplastic transformation of CGL1 cells. Loss of chromosome 11 alone was, therefore, found to be necessary but not sufficient for neoplastic transformation. We postulated that the loss of chromosome 11 may make the hybrid cells more susceptible to radiation-induced neoplastic transformation, since these cells have already undergone one of the required tumor suppressor loss events. Hybrid cells which have lost one copy of chromosome 11 were designated CON104(-11). CON104(-11) hybrid cells were found to have increased X-ray sensitivity and susceptibility to radiation-induced neoplastic transformation when compared with the parental CGL1 cells. In addition, the neoplastically transformed foci appear to arise earlier after radiation exposure in CON104(-11) versus CGL1 cells. Furthermore, the plating efficiency (PE) of the progeny of the irradiated CON104(-11) cells, growing in transformation flasks, is persistently lower than parental CGL1 cells during the 21 day assay period. The lower PE of the progeny of irradiated cells was attributed to the expression of delayed death/lethal mutations post-irradiation, a reflection of genomic instability. Taken together, the data indicate that previous loss of chromosome 11 may increase the radiation-induced genomic instability of the hybrid cells, leading to increased radiation sensitivity and neoplastic transformation potential. The data suggest that one possible function of the chromosome 11 tumor suppressor gene may be to help maintain genome stability after radiation damage.

Delayed apoptotic responses associated with radiation-induced neoplastic transformation of human hybrid cells.

HeLa X human skin fibroblast hybrid cells have been developed into a model for radiation-induced neoplastic transformation of human cells. Previous studies indicate that the appearance of neoplastically transformed foci in this system is delayed for several population doublings after irradiation and appears to involve the loss of putative tumor suppressor loci on fibroblast chromosomes 11 and 14. We now show that after treatment with 7 Gy of X-rays, transformed foci initiation correlates with delayed apoptosis initiated in the progeny of the irradiated cells after 10-12 cell divisions and with reduced plating efficiency (delayed death). The cells develop classic apoptotic morphology, positive terminal deoxynucleotidyl transferase-mediated nick end labeling and phosphatidylserine (annexin V) staining, and cleavage of poly(ADP-ribose) polymerase. In addition, a delayed induction of the p53 protein and the proapoptotic Bax protein is evident over a week after radiation exposure. We propose that a delayed build-up of mitosis-dependent genomic DNA damage or a loss of genetic material over time (10-12 cell divisions postirradiation) has two relevant outcomes: (a) cell death due to the delayed induction of a p53-dependent apoptosis; and (b) neoplastic transformation of a minor subset of survivors that has lost fibroblast chromosomes 11 and 14 (tumor suppressor loci for this system) and has either evaded apoptosis or not acquired enough genetic damage to induce apoptosis. It is postulated that both phenomena result from X-ray-induced, translesion-mediated genomic instability.

Molecular analyses of adaptive survival responses (ASRs): role of ASRs in radiotherapy.

Adaptive survival responses (ASRs), whereby cells demonstrate a survival advantage when exposed to very low doses of ionizing radiation (IR) 4 - 24 h prior to a high dose challenge, were first reported over 15 years ago. These responses were linked to hormesis, which implied that exposure to low levels of IR may be beneficial to the cell. We postulate that increased survival does not necessarily mean that the treatment is beneficial. Studies at the molecular level indicate that ASRs are the result of misregulated cell cycle checkpoint responses, occurring in the G1 phase of the cell cycle after IR. Specific gene products (i.e., PCNA, cyclin D1, cyclin A, XIP8, xip5 and xip13) appear to control these cell cycle checkpoint responses. Certain neoplastic cells show potent ASRs because they bypass checkpoints which would otherwise lead to apoptosis or other forms of cell death (possibly necrosis), and/or these cancer cells lack genetic factors, such as specific caspases (cysteine aspartate-specific proteases), that control apoptosis. Alterations in these cell cycle checkpoints or apoptotic responses may also occur during IR-induced stress responses in normal cells, at critical times (10-18 days posttreatment) following IR. One IR-induced protein, XIP8, may be a critical controlling factor at this point where delayed-onset apoptosis occurs. Additionally, we have shown that the presence or absence (i.e., SCID cells) of nonhomologous DNA double strand break repair did not seem to influence ASRs, suggesting that ASRs may be caused by signal transduction stress responses. ASRs may be beneficial to survival, however, the consequence(s) of that survival may be dire. For example, many neoplastic cells exhibited far greater ASRs than normal cells. Additionally, ASRs were induced by as little as 1 cGy and and were enhanced by repeated exposures of low level radiation. The implications for radiotherapy are that when a patient arrives for port film imaging during the course of therapy, the dose-rate, overall level of exposure, and time between port film exposure and high dose IR treatment become potentially important factors for improved efficacy of treatment of certain cancers. Further research is warranted to determine what molecular factors are most important for ASRs, and current work is focusing on XIP8.

Loss of suppressor loci on chromosomes 11 and 14 may be required for radiation-induced neoplastic transformation of HeLa x skin fibroblast human cell hybrids.

We have previously reported a linkage between radiation-induced damage to a putative tumor suppressor locus on fibroblast chromosome 11 and the re-expression of tumorigenicity in a hybrid cell line (HeLa x human skin fibroblast) used to study neoplastic transformation. Further investigation into the molecular basis of radiation-induced neoplastic transformation of the hybrid cell, CGL1, indicates that loss of fibroblast chromosome 11 appears to be necessary but not sufficient for neoplastic transformation. Previous analysis had suggested, though not clearly demonstrated, a possible role for loss of alleles on fibroblast chromosome 14 in the neoplastic transformation of the hybrid cells. Therefore, the status of chromosome 14 in the gamma-ray-induced, neoplastically transformed (GIM) hybrid cell lines and in nontumorigenic control (CON) hybrid cell lines isolated from irradiated populations has been investigated. Chromosome painting and molecular studies using restriction fragment length polymorphisms and tetranucleotide repeat polymorphism analysis were performed. As an additional control, the status of chromosome 12 was also examined. We report that five of the eight GIM cell lines have lost one complete copy of a fibroblast chromosome 14 while only one of the five CON cell lines has lost a complete copy of a fibroblast chromosome 14. No evidence of large-scale loss of chromosome 12 was detected in the GIM or CON cells. The data further suggest that both copies of fibroblast chromosome 14 contain an active tumor suppressor locus and that radiation-induced loss of either fibroblast chromosome 14 is associated with neoplastic transformation in this system. We now conclude that loss of alleles on both fibroblast chromosome 11 and 14 may be required for the radiation-induced neoplastic transformation of these human hybrid cells.

Evidence for a role of delayed death and genomic instability in radiation-induced neoplastic transformation of human hybrid cells.

HeLa x skin fibroblast human hybrid cells have been developed into a model of radiation-induced neoplastic transformation. The authors' studies indicate that the loss of putative tumour suppressor loci on fibroblast chromosomes 11 and 14 is evident after radiation-induced neoplastic transformation. How these fibroblast chromosomes/putative tumour suppressor loci are lost after radiation exposure is currently being investigated. It has been shown that the appearance of transformed foci correlates with the onset of the delayed reduction in plating efficiency or delayed death. This delayed death appears to be the result of the onset of a novel delayed apoptosis in the irradiated progeny beginning around day 8 post-irradiation. It was proposed that the reduction in plating efficiency and subsequent neoplastic transformation are all the result of a radiation-induced genomic instability. The instability process has two relevant outcomes: (1) cell death due to the induction of a delayed apoptosis in cells; and (2) neoplastic transformation of a small subset of survivors that have lost fibroblast chromosomes 11 and 14 (tumour suppressor loci) but either have not acquired enough genetic damage to induce the apoptotic response or have undergone molecular changes allowing them to bypass apoptosis. Data from the genomic instability and delayed death literature will be reviewed in terms of relevance to radiation-induced neoplastic transformation. New data are presented which demonstrate that use of growth media supplemented with a specific lot of calf serum was found to increase the number of cells undergoing radiation-induced neoplastic transformation, compared with standard serum after a fixed dose of radiation. This correlates with an increase in delayed death in the irradiated progeny which the authors propose is the result of increased genomic instability post-irradiation of cells grown in this serum. Preliminary data are presented indicating that a delayed apoptosis is also seen after high-energy He- particle exposure in this system.

In vitro cytotoxicity of silver-impregnated collagen cuffs designed to decrease infection in tunneled catheters.

PURPOSE: To examine in vitro the effects of silver-impregnated collagen cuff material from central venous catheters on human fibroblast growth. MATERIALS AND METHODS: In culture flasks, hybrid cells were exposed to silver-impregnated collagen cuff material, and human fibroblasts were exposed to silver-impregnated or silver-free collagen cuff material. After 72 hours of growth, cells were stained and digitally imaged, and the relative areas of cytotoxicity were determined. RESULTS: Flasks containing the silver-impregnated collagen cuff material and hybrid cells or human fibroblasts showed a marked local cytotoxic effect of the cuff material; cell-free zones surrounding the cuff material were demonstrated. No cytotoxic effect was seen in the flasks that contained silver-free cuff material (control group). Mean area of cleared cells was 312 mm2 +/- 130 (range, 156-624 mm2) in the flasks containing human fibroblasts and silver-impregnated cuff material and 0 mm2 in the corresponding control flasks (P < .0001). Mean radius of the area of cleared cells around the silver-impregnated cuff material in the flasks containing human fibroblasts was 9.8 mm +/- 2.0 (range, 7.0-14.1 mm). CONCLUSION: Silver-impregnated collagen cuff material demonstrates a local cytotoxicity on hybrid cells and human fibroblasts in vitro. This finding may explain the phenomena seen clinically of decreased anchorage and inadvertent removal of catheters with silver-impregnated collagen cuffs.

Loss of a putative tumor suppressor locus after gamma-ray-induced neoplastic transformation of HeLa x skin fibroblast human cell hybrids.

The nontumorigenic HeLa x skin fibroblast hybrid cell line, CGL1, can be induced to re-express HeLa tumor-associated cell surface antigen, p75-IAP (intestinal alkaline phosphatase), with resulting neoplastic transformation, by exposure to gamma radiation. This has allowed the human hybrid system to be developed into a quantitative in vitro model for radiation-induced neoplastic transformation of human cells. Recently, several gamma-ray-induced IAP-expressing mutants (GIMs) of the nontumorigenic HeLa x skin fibroblast hybrid CGL1 were isolated and all were tumorigenic when injected subcutaneously into nude mice (Mendonca et al., Cancer Res. 51, 4455-4462, 1991). Control cell lines which were negative for p75-IAP (CONs) were also isolated from irradiated populations, and none were found to be tumorigenic. We have now begun to investigate the molecular basis of radiation-induced neoplastic transformation in this system by studying the potential genetic linkage between p75/IAP expression, tumorigenicity and damage to a putative tumor suppressor locus on fibroblast chromosome 11. Previous analysis of rare spontaneous segregants has indicated that this locus is involved in the regulation of tumorigenicity and in the expression of the HeLa tumor-associated cell surface marker intestinal alkaline phosphatase (p75-IAP) in this system. Therefore, analysis by restriction fragment length polymorphism and chromosome painting have been performed for chromosome 11, and for chromosome 13 as a control, for the p75/IAP-positive GIM and p75/IAP-negative CON cell lines. We report that in five of eight of the GIMs large-scale damage to the fibroblast chromosome 11's is evident (four GIMs have lost one complete copy of a fibroblast chromosome 11 and one GIM has both copies of fibroblast chromosome 11 heavily damaged). None of the CONs, however (0/5), have lost a complete copy of either fibroblast chromosome 11. No large-scale damage to the control chromosome 13's was detected in the GIMs or CONs. The data further suggest that both copies of fibroblast chromosome 11 contain an active locus and that radiation-induced loss of either fibroblast chromosome 11 will result in neoplastic transformation in this system. We conclude that it is the loss of a putative tumor suppressor locus on fibroblast chromosome 11 which is responsible at least in part for radiation-induced neoplastic transformation of these human hybrid cells.

The effect of postirradiation holding at 22 degrees C on the repair of sublethal, potentially lethal and potentially neoplastic transforming damage in gamma-irradiated HeLa x skin fibroblast human hybrid cells.

The effect of postirradiation holding at 22 degrees C on cell growth, progression of cells through the cell cycle, and the repair of sublethal, potentially lethal and potentially neoplastic transforming damage in gamma-irradiated HeLa x skin fibroblast human hybrid cells has been examined. Cell growth and cell cycle progression were essentially stopped at this reduced temperature. Cell survival was dramatically reduced by holding confluent cultures for 6 h at 22 degrees C, as opposed to 37 degrees C, after 7.5 Gy gamma radiation delivered at a rate of 2 Gy/min. Return of the cells to 37 degrees C for 6 h after holding at 22 degrees C did not result in increased survival. A similar effect was obtained when the cells were held at 22 degrees C between split-dose irradiation of log-phase cultures where no increase in survival was observed over a split-dose interval of 4 h. In this case a partial increase in survival was observed upon returning the cells to 37 degrees C for 3 h after holding at 22 degrees C for the first 3 h of the split-dose interval. Neoplastic transformation frequency was not enhanced by holding confluent cultures for 6 h at 22 degrees C after 7.5 Gy gamma radiation. This is consistent with previous observations that misrepair of potentially neoplastic transforming damage already occurs at 37 degrees C. The overall results are interpreted in terms of the reduced temperature favoring misrepair, rather than inhibition of repair, of sublethal, potentially lethal and potentially transforming radiation damage.

Delayed heritable damage and epigenetics in radiation-induced neoplastic transformation of human hybrid cells.

The HeLa x skin fibroblast human hybrid cell system has proven to be an excellent model system for quantitative studies of radiation-induced neoplastic transformation in vitro. A unique aspect of this system is the reexpression of a cell surface protein p75/150 with tumorigenicity. The identification of p75/150 as intestinal alkaline phosphatase (IAP) allowed for the recent development of a more simplified, rapid, and sensitive screening method than the previous p75/150 antibody-based staining procedure. The new method directly detects neoplastically transformed, IAP-expressing cells by staining with the alkaline phosphatase chromogenic substrate, Western Blue (WB). Earlier studies with the antibody-based immunoperoxidase assay indicated that, while no foci with tumor-associated antigen (p75-positive) were evident 15 days after irradiation, the number of foci rose quickly and leveled off between Day 19 and Day 23. This late appearance of the IAP-positive foci suggested that the neoplastic transformation process was not an immediate consequence of radiation damage. The mechanism underlying this observation was unknown. The possibility existed that very small foci and/or foci expressing a low level of IAP were being missed at earlier expression times. The increased sensitivity of the WB staining technique has allowed for the reinvestigation of the kinetics of induction of radiation-induced foci in this system. Experiments were performed where parallel groups of transformation flasks were stained at Days 7, 9, 11, 13, 15, 17, 19, and 21 days after irradiation. The data clearly indicate that the radiation induction of IAP-positive foci is indeed delayed in this system with the vast majority of the foci beginning to appear after Day 9 after irradiation. The delay is not the result of a lack of ability to detect small IAP-positive foci since foci with as few as 15 IAP-positive cells were discernible. We have reported previously that under identical experimental conditions both the establishment of plateau phase and the onset of the expression of lethal mutations also occur after Day 9. We therefore propose that radiation-induced neoplastic transformation of HeLa x skin fibroblast hybrid cells is a consequence of the delayed expression of heritable damage under epigenetic control with a resultant loss of tumor-suppressor function.