•3′-5′ ssExos are essential for recB1080 mutant, unlike in wt, recD and recB strains.•3′-5′ ssExos act in synergy with RecQ helicase in recB1080 mutant.•3′-5′ ssExos and RecQ act on a long and ...reactive 3′ tail devoid of RecA protein.•3′ tail metabolism is as important as elimination of 5′ tail for DNA-end resection.
Degradation of a 5′-ending strand is the hallmark of the universal process of DNA double strand break (DSB) resection, which results in creation of the central recombination intermediate, a 3′-ending overhang. Here we show that in Escherichia coli recB1080/recB1067 mutants, which are devoid of RecBCD's nuclease and RecA loading activities, degradation of the unwound 3′ tail is as essential as is degradation of its 5′-ending complement. Namely, a synergistic action of ExoI, ExoVII, SbcCD and ExoX single-strand specific exonucleases (ssExos) of 3′-5′ polarity was essential for preserving cell viability, DNA repair and homologous recombination in the recB1080/recB1067 mutants, to the same extent as the redundant action of 5′-tail trimming ssExos RecJ and ExoVII. recB1080 derivatives lacking 3′-5′ ssExos also showed a strong induction of the SOS response and greatly increased SOS-dependent mutagenesis. Furthermore, we show that ExoI and ExoVII ssExos act synergistically in suppressing illegitimate recombination in the recB1080 mutant but not in a wt strain, while working in concert with the RecQ helicase. Remarkably, 3′-5′ ssExos show synergism with RecQ helicase in the recB1080 mutant in all the assays tested. The effect of inactivation of 3′-5′ ssExos in the recB1080/recB1067 mutants was much stronger than in wt, recD, and recB strains. These results demonstrate that the presence of a long, reactive 3′ overhang can be as toxic for a cell as its complete absence, i.e. it may prevent DSB repair. Our results indicate that coupling of helicase and RecA-loading activity during dsDNA-end resection is crucial in avoiding the deleterious effects of a long and stabile 3′ tail in E. coli.
Double-strand breaks (DSBs) are lethal DNA lesions, which are repaired by homologous recombination in
To study DSB processing
, we induced DSBs into the
chromosome by γ-irradiation and measured ...chromosomal degradation. We show that the DNA degradation is regulated by RecA protein concentration and its rate of association with single-stranded DNA (ssDNA). RecA decreased DNA degradation in wild-type,
, and
strains, indicating that it is a general phenomenon in
On the other hand, DNA degradation was greatly reduced and unaffected by RecA in the
mutant (which produces long overhangs) and in a strain devoid of four exonucleases that degrade a 3' tail (ssExos). 3'-5' ssExos deficiency is epistatic to RecA deficiency concerning DNA degradation, suggesting that bound RecA is shielding the 3' tail from degradation by 3'-5' ssExos. Since 3' tail preservation is common to all these situations, we infer that RecA polymerization constitutes a subset of mechanisms for preserving the integrity of 3' tails emanating from DSBs, along with 3' tail's massive length, or prevention of their degradation by inactivation of 3'-5' ssExos. Thus, we conclude that 3' overhangs are crucial in controlling the extent of DSB processing in
This study suggests a regulatory mechanism for DSB processing in
, wherein 3' tails impose a negative feedback loop on DSB processing reactions, specifically on helicase reloading onto dsDNA ends.
The role of T regulatory lymphocytes (T
) particularly in cancer is well known. The goal of the present study was to determine the contribution of these lymphocytes in the regulation of anti-tumor ...immunity of CBA/HZgr mice against MC-2 fibrosarcoma (4
generation of methylcholanthrene induced tumor). The levels of T lymphocytes (CD4+, CD8+ and CD4+CD25+) were determined 8 and 20 days after tumor transplantation. Further, the role of CD4+CD25+ (T
) in tumor-host interaction was evaluated
and
by using specific monoclonal antibodies. We found that splenocytes of both control and T
depleted tumor bearing mice strongly but differently inhibited growth of tumor cells
. While splenocytes of untreated mice exhibited significant decrease of this activity (from 74.4% to 62.6% and 32.95%), the splenocytes of T
depleted mice showed increase of this activity (from 79.5% to 84.3% and 86.2%) from day 6 to day 13 and day 21 after tumor grafting, respectively. Further, upon i.v. injecting specific monoclonal anti-T
antibody tumor immediately prior to tumor cell intracutaneous transplantation, the tumor was rejected after initial growth. In treated mice, the incidence of T
cells was very low initially, reaching normal values two weeks later. These animals were shown to be resistant to tumor transplantation four months later.
The dynamics of SCCVII transplantable tumor growth in C3H/H mice was determined after local tumor irradiation and/or virus (NDV LaSota) i.p. injection. The virus applied alone significantly ...suppressed tumor growth, particularly until the 19th day after tumor transplantation. Local irradiation with 30 Gy resulted in tumor disappearance followed with its regrowth about 15 days later. However, if the virus was injected after the irradiation, there was no tumor growth until the end of the 31 day observation period. It should be noted that virus application prior to local irradiation did not have any additional influence on tumor growth. Thus, the pronounced efficacy of virus applied after tumor irradiation deserves attention. It is possible that the virus injected after irradiation induced a chain of cytokine production joining the action of tumor destruction induced by irradiation. This should be further studied in clarifying the approaches to combined tumor therapy with possible cell-free vaccine production.
The cytotoxic activity of phenylboroxine acid was evaluated in vitro on mouse mammary adenocarcinoma 4T1, mouse squamous cell carcinoma SCCVII, hamster lung fibroblast V79 and mouse dermal ...fibroblasts L929 cell lines. The cytotoxic effects were dose dependent for all tested tumour and non-tumour cell lines. Under in vivo conditions, three application routes of phenylboronic acid were studied: intra-peritoneal (i.p.), intra-tumour (i.t.) and per-oral. After tumour transplantation in syngeneic mice, phenylboronic acid was shown to slow the growth of both tumour cell lines (4T1 and SCCVII) compared with the control. The inhibitory effects were pronounced during the application of phenylboronic acid. For both tested tumour cell lines, the most prominent antitumour effect was obtained by intraperitoneal administration, followed significantly by oral administration.
C3Hf/HZgr mice were transplanted with SCCVII carcinoma cells and treated with Newcastle disease virus (NDV). The treatment slows down the growth of transplanted tumor. Furthermore, by using specific ...monoclonal antibodies, the frequencies of CD4+, CD8+, and CD4+CD25+ T lymphocytes were determined in the spleen of tumorous mice at particular times following tumor transplantation and/or NDV application. The incidence of lymphocytes CD4+ and CD8+ decreased and of CD4+CD25+ increased in the spleen of mice during the time following tumor transplantation. However, the frequency of regulatory CD4+CD25+ T lymphocytes in the spleen is very low, while CD4+ and CD8+ increased to normal level following intraperitoneal (i.p.) NDV injection in tumor-bearing mice. Thus, besides directly destroying transplanted tumor, NDV seems to be involved against growing tumor by reducing the frequency of regulatory T lymphocytes maintaining the frequency of CD4+ and CD8+ T lymphocytes within the control values pointing to its role in immunomodulation.
Najčešći i uobičajeni pristupi u liječenju tumorskih bolesti uključuju kirurgiju, radioterapiju, kemoterapiju, imunoterapiju, ciljanu terapiju, hormonsku terapiju, transplantaciju matičnih stanica te ...sveobuhvatno personalizirano liječenje. Izostavlja se gotovo zaboravljeno liječenje primjenom različitih mikroorganizama od kojih su najzastupljeniji virusi. Njihova je neposredna primjena, ako se radi o apatogenim virusima ili njihovim genomskim modifikacijama, sve učestalija i postigla je vrlo visok stupanj primjenjivosti. Ovaj prikaz odnosi se pretežno na mogućnosti primjene mikroorganizama, u manjoj mjeri bakterija, a uglavnom virusa, u liječenju tumorskih bolesti. Neki virusi mogu zaraziti i ubiti tumorske stanice te su poznati kao onkolitički. Neki se od njih nalaze u prirodi, ali su također i modificirani, kako bi se pojačalo njihovo onkolitičko svojstvo, djelujući isključivo na tumorske stanice bez oštećivanja zdravih. U najnovije vrijeme genetički modificirani virusi postigli su stupanj javne upotrebe stavljanjem na tržište te su od FDA-a (Food and Drug Administration) dopušteni za liječenje melanoma (T-VEC)¸ dok se u Kini modificiranim adenovirusom (H101) liječi rak glave i vrata ljudi. Posebna je vrijednost virusa u liječenju tumorskih bolesti, jer se umnažaju u tumorskoj stanici dok je ne razore, tvoreći tako kompleks tumorskog antigena što omogućuje njegovo prepoznavanje od strane imunosnog sustava. Reakcija tog sustava je lokalna ili sustavna, prepoznata danas kao imunoterapija. Uspoređeni s klasičnim oblicima liječenja tumorskih bolesti, primjena onkolitičkih virusa i imunoterapija uspješniji su način s tek blagim nuspojavama. Danas suvereno usmjeruju istraživanja upravo u tom smislu.
Two kinds of nanosize V
2O
5 particles were synthesized in our own laboratory and concomitantly applied to V79 and L929 fibroblasts and SCCVII, B16F10 and FsaR tumor cells. The morphologies of the ...cells were monitored using an inverted inverse microscope equipped with digital camera, while quantitative determination of the cytotoxicity of nanosize V
2O
5 particles was measured using crystal violet bioassay. Twenty four hours after the addition of nanosize V
2O
5 particles (20
μM), noticeable changes in the morphology and density of fibroblast and cancer cells were observed. Reculturing in a freshly prepared medium for the next 24
h showed a high recovery effect on V79, SCCVII and B16F10 cells, while FsaR and L929 cells were seriously damaged and unable to recover. At a higher concentration of nanosize V
2O
5 particles (100
μM), the cytotoxicity of V
2O
5 prevailed against the recovery effect in all cell types. Quantitative measurements have shown that the resistance of investigated cell cultures to the cytotoxicity of nanosize V
2O
5 particles decreases in the order V79
>
SCCVII
>
B16F10
>
FsaR
>
L929. The high cytotoxic effect found on FsaR cells suggests that nanosize V
2O
5 particles could be regarded as poisoning material in the treatment of FsaR fibrosarcoma cells. Possible mechanisms involved in the cytotoxicity of nanosize V
2O
5 particles were discussed.
The aim of this study was to detect the antitumor properties of Croatian propolis in BALB/c male and female mice injected with 4T1 mammary carcinoma. Furthermore, the gender-dependence of this effect ...and the possible involvement of combined effect of propolis and 5-Fluorouracil (5FU) on dihydropyrimidine dehydrogenase (DPD) transcriptional and translational level, were determined. In combination with 5FU propolis treatment induced gender-related effects. The results of the study revealed that pretreatment of mice with propolis combined with 5FU treatment prolonged the suppressive effect of 5FU on tumor growth and reduced the number of metastasis only in male mice. Only males pretreated with propolis prior to 5FU administration had decreased DPD protein level indicating higher sensitivity to 5FU. Thus, benefitial effects of propolis in male tumor-bearing mice treated with 5FU might be explained by increased sensitivity to 5FU as the result of translationally downregulated DPD.
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