Optogenetic Control of Non-Apoptotic Cell Death.

Herein, a set of optogenetic tools (designated LiPOP) that enable photoswitchable necroptosis and pyroptosis in live cells with varying kinetics, is introduced. The LiPOP tools allow reconstruction of the key molecular steps involved in these two non-apoptotic cell death pathways by harnessing the power of light. Further, the use of LiPOPs coupled with upconversion nanoparticles or bioluminescence is demonstrated to achieve wireless optogenetic or chemo-optogenetic killing of cancer cells in multiple mouse tumor models. LiPOPs can trigger necroptotic and pyroptotic cell death in cultured prokaryotic or eukaryotic cells and in living animals, and set the stage for studying the role of non-apoptotic cell death pathways during microbial infection and anti-tumor immunity.

Crosstalk with cancer-associated fibroblasts increases the growth and radiation survival of cervical cancer cells.

Crosstalk between cancer cells and the surrounding cancer associated fibroblasts (CAFs) plays an illusive role in cancer radiotherapy. This study investigated the effect of cancer cell-cancer associated fibroblasts crosstalk on the proliferation and survival of irradiated cervical cancer cells. A pretreatment with conditioned medium from a mixed culture of CAF and HeLa cells (mixCAF) had a stronger effect on enhancing the proliferation and survival of irradiated HeLa cells compared to pretreatment with CAF conditioned medium alone. In addition, pretreatment with a mixed culture of CAF and HeLa cells conditioned medium reduced the levels of two major radiation-induced genes, GADD45 and BTG2, and phosphorylation of p38. Profiling of the growth and survival factors in the conditioned medium revealed PDGF and VEGF, and IGF2, EGF, FGF-4, IGFBPs and GM-CSF to be specifically secreted from HeLa cells and CAFs, respectively. This study demonstrated radiation protective effects of CAF-cancer cell crosstalk, and identified multiple growth factors and radiation response genes that might be involved in these effects.

Recombinant human endostatin, Endostar, enhances the effects of chemo-radiotherapy in a mouse cervical cancer xenograft model.

BACKGROUND: The effects of recombinant human endostatin, Endostar, combined with concurrent chemo-radiotherapy (CCRT) on tumor growth, angiogenesis and lymphangiogenesis in a mouse xenograft model of cervical cancer was investigated. METHODS: HeLa cells were injected subcutaneously to establish mouse xenograft models and mice were treated with normal saline (control), CCRT with cisplatin (CDDP), Endostar, or a combination of Endostar and CCRT. Growth, metastasis, and angiogenesis of tumors was monitored. RESULTS: Tumorogenic activity of tumor cells in the CCRT, Endostar and combination Endostar-CCRT treatment groups was markedly decreased compared with the activity in the NS group (p < 0.05). The most significant inhibition of tumor growth was observed in the Endostar with CCRT group. Lymph node metastases in the Endostar with CCRT group (12.5%) and Endostar alone group (25%) were lower compared to the CCRT group (42.8%) and NS group (66.7%; p < 0.05). Endostar was also found to inhibit tumor angiogenesis. Endostar induced apoptosis of HeLa cells in vivo, and inhibited expression of VEGF and HIF-1alpha in vivo and in vitro. CONCLUSION: Endostar enhanced the anti-cancer effect of CCRT in a mouse xenograft model of cervical cancer. These findings thus provide a new strategy to treat cervical cancer.

[Requiem for Henrietta]. / Requiem pour Henrietta.

Fifty years after Henrietta Lacks died of aggressive glandular cervical cancer, the first cell line - HeLa cell line - is the workhorse of laboratories everywhere. It helped to produce drugs for numerous diseases, including poliomyelitis, Parkinson's, leukemias. But they are so outrageously robust that they contaminated hundred of other cell lines, as far away as Russia. For decades, biologists worked with contaminated cell lines and today, the problem is not yet solved. But the story of HeLa cells is also a moving reflection of racial and ethical issues in medicine in the late half-twentieth century in the USA.

Poly(ethylene glycol)-grafted polyethylenimine modified with G250 monoclonal antibody for tumor gene therapy.

To improve the biocompatibility of a gene vector and to avoid its being eliminated by the immune system, polyethylenimine (PEI) was modified with poly(ethylene glycol) (PEG) before G250 monoclonal antibody (mAb) conjugation. G250-PEI-PEG was capable of forming complexes with DNA in the right size distribution, and the G250 mAb modification significantly improved PEI transfection of G250-positive cells. The highest transfection efficiency was seen in HeLa cells as determined by flow cytometry after transfection with the gene encoding green fluorescent protein: 2-fold higher compared with the transfection of HepG2 cells. Blocking the surface antigen on the cell membrane of HeLa cells by incubation with free G250 mAb, or by downregulating G250 expression by small interfering RNA transfection, resulted in a remarkable decrease in transfection efficiency. These data indicate the targeting effect of G250 antibody modification. The presence of serum decreased transfection efficiency in a concentration-dependent manner. However, the transfection of HeLa cells with G250-PEI-PEG remained significant in the presence of 30% serum. In an in vivo study, G250-PEI-PEG exhibited high transfection efficiency in tumors. In addition, pathological analysis did not show obvious toxicity caused by the materials used. These suggest that PEG- and G250 mAb-modified PEI could be a useful nonviral gene vector for in vivo study.

Higher susceptibility of NOG mice to xenotransplanted tumors.

The purpose of tumorigenicity testing, as applied not only to cell substrates used for viral vaccine manufacture but also stem cells used for cell-based therapy, is to discriminate between cells that have the capacity to form tumors and cells that do not. Therefore, tumorigenicity testing is essential in assessing the safety of these biological materials. Recently developed NOD/Shi-scid IL2Rg(null) (NOG) mice have been shown to be superior to NOD/Shi-scid (SCID) mice for xenotransplantation of both normal and cancerous cells. To select a suitable mouse strain as a xenogenic host for tumorigenicity testing, we compared the susceptibility of NOG (T, B, and NK cell-defective), SCID (T and B cell-defective), and the traditionally used nude (T cell-defective) mice to tumor formation from xenotransplanted HeLa S3 cells. When 10(4) HeLa S3 cells were subcutaneously inoculated into the flanks of these mice, the tumor incidence on day 22 was 10/10 (100%) in NOG, 2/10 (20%) in SCID, and 0/10 (0%) in nude mice. The subcutaneous tumors formed reproducibly and semiquantitatively in a dose-dependent manner. Unexpectedly, half of the NOG mice (5/10) that had been inoculated with a mere 10(1) HeLa S3 cells formed progressively growing subcutaneous tumors on day 78. We confirmed that the engrafted tumors originated from inoculated HeLa S3 cells by immunohistochemical staining with anti-HLA antibodies. These data suggest that NOG mice may be the best choice as a suitable strain for testing tumorigenicity.

SOCS1 [corrected] inhibits HPV-E7-mediated transformation by inducing degradation of E7 protein.

Human papilloma viruses (HPVs) are small double-stranded DNA viruses that infect mucosal and cutaneous epithelium and induce cervical cancer. It has been shown that interferon (IFN)gamma suppresses proliferation of HPV-infected cells by suppressing expression of HPV E7. Here, we found that IFNgamma induces not only suppression of E7 transcription but also proteasome-dependent degradation. Suppressor of cytokine signaling-1 (SOCS1)/JAB, a suppressor of cytokine signaling, is known to be induced by IFNgamma, and functions as an antioncogene against various hematopoietic oncogenic proteins. SOCS1 contains the SOCS-box, which is shown to recruit ubiquitin transferase to the molecules that interact with SOCS1. We found that SOCS1 interacted with HPV E7 protein and induced ubiquitination and degradation of E7 in a SOCS-box-dependent manner. SOCS1 overexpression also increased Rb protein levels and suppressed proliferation of cervical cancer cell lines infected with HPV. Moreover, E7 protein levels were higher and Rb protein levels were lower in SOCS1-deficient fibroblasts infected with retrovirus vector carrying E7 gene than in wild-type fibroblasts. E7 induced anchorage-independent growth in SOCS1-deficient fibroblasts, but not in wild-type cells. These data suggested that SOCS1 plays an important role in regulating the levels of E7 protein and their transforming potential, and could be a new therapeutic tool for HPV-mediated tumors.

Combined low dose radio- and radioimmunotherapy of experimental HeLa Hep 2 tumours.

Radiation therapy of malignant tumours can be delivered by external beam radiation (RT) or radioimmunotherapy (RIT), using nuclides attached to monoclonal antibodies (mAbs). These treatment modalities have now been combined in order to investigate putative therapeutic advantages and elucidate the biological responses involved. Nude mice were transplanted subcutaneously on the back with human HeLa Hep2 tumour cells. RT (3x5 Gy) and/or 100 microg (131)I-labelled mAb H7, against placental alkaline phosphatase, or (131)I-labelled mAb TS1, against cytokeratin, was administered separately or in combination (specific activity of 120-200 MBq/mg antibody). Significant tumour growth retardation was observed both with RT alone and with RIT alone. Combining these regimens enhanced the therapeutic effects further, and a significant reduction in tumour volume could be demonstrated. The tumours were subjected to extensive histochemical and immunohistochemical investigations in order to elucidate changes in biology and histology within them. The following stainings were used: haematoxylin-eosin (morphology), Ki67 (proliferation), M30 (apoptosis), TUNEL (apoptosis) and endoglin (vascularisation). Tumours in the control group grew fast, with an average tumour doubling time of 9 days. These tumours contained large viable tumour cell masses displaying vast proliferation zones of Ki67-positive tumour cells, as well as necrotic regions and small amounts of connective tissue. Apoptotic cells could be identified both with M30 and TUNEL staining. When RT was applied, the growth rate was significantly reduced (doubling time 19 days) and typical alterations in morphology were seen, with a relative increase in connective tissue and a decrease in necrotic regions. Apoptotic cells were identified and a decrease in cell density was also observed. When RIT alone was applied, the growth parameters indicated a longer lasting growth reduction, especially when TS1 was used separately or in combination with H7. The histological appearances of these tumours were somewhat different from the RT-treated tumours, with a larger portion of intratumoural cysts. These tumours also presented a reduced tumour cell density. Dramatic effects were observed when RT was combined with RIT, with a pronounced growth reduction seen in all combination treatment groups. Pronounced tumour volume reduction was also evident in both the RT + RIT ((131)I-TS1) group and RT + RIT ((131)I-TS1/(131)I-H7) group, and in some animals no tumour remained at all. The morphology of the tumour remnants at day 22 was chaotic with a drastically changed histology, with presence of abundant cysts, low fractions of Ki67-positive cells, reduction in cell density, increased amounts of connective tissue and a decrease in necrotic regions. Again, apoptotic cells could be identified, scattered throughout the viable regions. Combining RT and RIT seems to generate an efficient treatment with convincing and long-lasting tumour growth inhibition, which is reflected in a highly aberrant histology within the tumour. Results obtained in this study indicate that both necrosis and apoptosis may be involved in the process leading to this efficient therapy of epithelially derived tumours.

BMP-4 and BMP-6 involvement in the osteogenic properties of the HeLa cell line.

The heterotopically induced ossicles are used in our research on bone tissue. The ossicles are formed in the thigh muscle of BALB/c mice under the influence of injected suspension of 3 x 10(6) HeLa cells. We postulate that the mechanism of bone induction is based on the secretion of bone morphogenetic proteins BMP-4 and BMP-6 by the grafted HeLa cells. This was proved by the use of specific immunohistochemical reaction and Western blots of conditioned culture medium. It seems that HeLa cells secrete BMPs continuously into the culture medium, even without contact with the mice muscle tissue, were induction of bone tissue is observed.

Production of recombinant H1 parvovirus stocks devoid of replication-competent viruses.

Vector and helper plasmids for the production of recombinant H1 (rH1) parvovirus, an oncolytic virus and candidate vector for cancer gene therapy, were constructed with the aim of reducing the contamination of these preparations with replication-competent viruses (RCV). Split-helper plasmids were constructed by manipulating the splicing signals for the capsid proteins such that VP1 and VP2 were expressed from separate plasmids. H1 vectors with similarly mutated splice sites were packaged, using the split-helper plasmids, and the resulting recombinant H1 viruses were completely free of RCV because the generation of recombinants expressing both capsid proteins was prevented. Vector yields of rH1 produced with split-helper plasmids in combination with splice site-modified vectors were similar (in the range of 10(7) replication units/ml) to yields of rH1 produced with the standard vector/helper pair, in which case significant levels of RCV were generated (10(4)-10(5) plaque-forming units/ml). To assess the functionality of this approach in vivo, rH1 was produced that contained the human interleukin 2 (IL-2) transgene and that was devoid of RCV. This IL-2-carrying rH1 vector expressed IL-2 efficiently in human tumor cells (HeLa) in vitro and generated antitumor responses in nude mice xenografted with HeLa cells that had been infected ex vivo with this virus. These results should allow the large-scale production of recombinant oncotropic parvoviruses and their assessment for the gene therapy of cancer in a clinical setting.

Correlation between growth control, neoplastic potential and endogenous connexin43 expression in HeLa cell lines: implications for tumor progression.

A HeLa cell line, obtained from the ATCC, was cloned and found to exhibit a spectrum of in vitro and in vivo growth characteristics as well as variable expression of endogenous connexin43 (Cx43), a widely expressed gap junction protein implicated in growth control. The majority of clones expressed functional Cx43, which contrasted with previous studies reporting that HeLa cells are completely negative for Cx43 mRNA/protein expression. This endogenous Cx43 expression correlated with increased growth control: Cx43-positive clones exhibited a decreased saturation density and a diminished growth capacity when in co-culture with growth-controlled normal cells in constrast to Cx43-negative clones. Endogenous Cx43 expression was negatively correlated with neoplastic potential as evidenced by attenuated anchorage-independent growth and decreased tumorigenicity in immunodeficient mice. Treatment of Cx43-negative cells with 5-aza-2'-deoxycytidine resulted in expression of Cx43, suggesting gene silencing via DNA methylation. These results support the concept of growth control via junctionally transmitted signals and suggest an epigenetic mechanism for tumor cells to circumvent this control during carcinogenesis. Moreover, the heterogeneous nature of this cell line and the ease of connexin43 gene induction suggest caution in the interpretation of results involving gene transfection using noninducible gene expression systems.

Pathophysiological effects of human TNF-alpha-producing tumor xenografts in immunosuppressed mice.

Groups of CBA mice immunosuppressed with anti-thymocyte serum (ATS) treatment were xeno-transplanted with either HeLa human cervical carcinoma cells or genetically modified cells expressing the human tumor necrosis factor-alpha (TNF) gene (All cells). Both cell lines were highly resistant to the cytotoxic effects of TNF. If 3 x 10(6) tumor cells were inoculated s.c. into female mice, HeLa cells grew progressively into large tumors and killed 74% of the recipients, while TNF-expressing All cells caused fatal tumor growth only in 22% of the mice. 3 x 10(6) or 1.5 x 10(7). All cells produced progressive tumor growth and lethality in all male recipients. In sera of all the A11-cell-transplanted mice, biologically active TNF was detected shortly (4.5 h) after tumor inoculation (6 39 U/ml), decreasing to below detection level in the circulation by day 3. In recipients of 15 million A11 cells, circulating TNF reappeared and reached high levels (12-1000 U/ml) 3 to 7 weeks later, when the animals bore large tumors (14-23 mm). Generally, such mice became cachectic, severely anemic, hypothermic, and soon died. On account of calcium mobilization from bones, their serum Ca levels were high. Electron microscopy revealed severe liver damage, but there were no signs of chronic arthritis. These results suggest that ATS-treated mice xenotransplanted with TNF-gene-transfected A11 human tumor cells provide a new model for studying the pathophysiological and anti-tumor effects of TNF.

Encapsulation in hollow fibres of xenogeneic cells engineered to secrete IL-4 or IL-13 ameliorates murine collagen-induced arthritis (CIA).

A strategy of gene therapy using IL-4 or IL-13 xenogeneic transfected cells encapsulated into permeable hollow fibres (HF) was used to treat CIA. Hydrogel-based hollow fibres were obtained from AN-69 copolymer, already known for its biocompatibility and tolerance in rodents. Permeability to IL-4 and lack of cell leakage from the fibres were ascertained in vitro and in vivo. Chinese hamster ovary (CHO) fibroblasts transfected with mouse IL-4 gene were encapsulated in HF (6.25 x 105 cells/HF). IL-4 was detected in vitro in the culture supernatant of filled fibres for at least 19 days. IL-4 or IL-13 transfected CHO cells encapsulated in HF were implanted in the peritoneum of mice on days 11-13 after immunization with type II collagen. Control mice were treated with fibre containing CHO cells transfected with beta-galactosidase (betagal) gene; a positive control group consisted of mice treated by subcutaneous injection of 106 cells on days 10 and 25. Mice were monitored for signs of arthritis by observers unaware of the status of animals. Results of these experiments indicate that severity of the articular disease was significantly reduced in the groups of mice treated with CHO/IL-4 or CHO/IL-13 cells encapsulated in HF, compared with control groups receiving CHO/betagal cells encapsulated in HF. Histological analysis confirmed these data and extended them to a better inhibitory effect of encapsulated cells compared with free cells on inflammatory and destructive joint disease. Moreover, such long-term treatment with HF was well tolerated; macroscopic and histological aspects of peritoneal cavity were moderately inflammatory. Thus, our results may have important implications for clinical use of gene transfected cells as therapeutic agents in the treatment of autoimmune diseases.

Suppression of malignancy by the 3' untranslated regions of ribonucleotide reductase R1 and R2 messenger RNAs.

Mammalian ribonucleotide reductase is rate limiting for the synthesis of DNA. The active enzyme is composed of two dissimilar components called R1 and R2, encoded by different genes. The 3' untranslated regions (3' UTRs) of R1 and R2 messages contain sequences that are important in regulating gene expression through changes in message stability. We have constructed expression plasmids containing the R1 or R2 mRNA 3' UTRs, and we show that transfection of these plasmids into highly malignant mouse 10 T1/2 cells significantly suppresses the tumorigenic properties of these cells in syngeneic mice when compared with cells transfected with the same plasmid lacking R1 or R2 3' UTR sequences or when compared with cells transfected with the same plasmid expressing a heterologous sequence as a control. Furthermore, cells expressing the R2 3' UTR exhibit significantly reduced potential to disseminate to the lungs of syngeneic animals in experimental metastasis assays. The tumor-suppressive effects of the mouse R1 and R2 3' UTRs were not confined to mouse cells, because human HeLa cells transfected with expression plasmids containing either RI or R2 3' UTRs were also significantly less tumorigenic in assays using BALB/c nu/nu mice. These studies demonstrate that the untranslated regions of ribonucleotide reductase mRNAs can function as modifiers of tumor cell development and for the more complex process of tumor dissemination. We propose that these malignancy-suppressive effects are mediated through RNA interactions with cellular components involved in growth regulation through mechanisms of posttranscriptional control of gene expression. In addition, these observations emphasize the enormous potential of untranslated RNA to act directly as modifiers of biological characteristics relevant to mechanisms of malignancy.

Biological activity assays of cell-free reassembled nuclei--injecting cell-free reassembled nuclei into unfertilized eggs can induce the eggs to cleave and reconstitute asters, and the injected nuclei undergo cell cycle changes.

Nucleus may reassemble spontaneously in cell-free mixture of HeLa metaphase chromosomes, Xenopus egg extracts and ATP-regenerating system, and the nucleus shows some biological activities. It is found that, after being injected into unfertilized mature eggs, the cell-free reassembled nuclei can cause the eggs to cleave and reconstitute asters in their cytoplasm, and the injected nuclei undergo changes in response to cell cycle regulators stored in the eggs, and that reinjecting cytostatic factors (CSF) into the eggs can stabilize the eggs in mitotic phase, cause the nuclei disassembly and chromatin condensation to chromosomes.

Identification of a human chromosome 11 gene which is differentially regulated in tumorigenic and nontumorigenic somatic cell hybrids of HeLa cells.

The tumorigenicity of HeLa cells in nude mice can be suppressed by the addition of a normal human chromosome 11 in somatic cell hybrids. We have attempted to identify specific genes involved in this phenomenon by transfecting a complementary DNA expression library into a tumorigenic HeLa-fibroblast hybrid. A cell line designated F2 was isolated which displayed morphological features of the nontumorigenic hybrids, demonstrated reduced tumorigenicity in nude mice, and showed an 85% reduction in alkaline phosphatase, a consistent marker of the tumorigenic phenotype in these cells. F2 contained a single exogenous complementary DNA, which was recovered by polymerase chain reaction and designated HTS1 because of its potential association with "HeLa tumor suppression." Northern blot studies suggested differential regulation of the HTS1 gene dependent on the tumorigenicity of the cell. In nontumorigenic hybrids, RNA species of 2.8, 3.1, and 4.6 kilobases were identified. In two tumorigenic hybrid lines, the 2.8-kilobase species was markedly reduced or absent. Similarly, three nontumorigenic human keratinocyte lines expressed all three RNA species, whereas several tumorigenic cervical carcinoma cell lines lacked the 2.8-kilobase species. Chromosome localization studies mapped the HTS1 gene to chromosome 11p15, a region of chromosome 11 that is believed to contain a tumor suppressor gene. These findings indicate that HTS1 represents a novel chromosome 11 gene which may be a target of the tumor suppressor gene active in this system.

A threshold effect in the induction of tumorigenicity of an established human cell line by v-mos.

The nontumorigenic immortal human cell line, SV80, was transfected with the v-mos gene to assess the gene's effect on tumorigenicity of cultured human cells. Two classes of cells, each containing functional v-mos, were obtained. The first class contained low levels of v-mos RNA, was morphologically transformed, but was nontumorigenic in nude mice. The second was also morphologically transformed, but contained high levels of v-mos RNA and was tumorigenic. The results indicate that SV80 cells behave similarly to murine fibroblasts in their response to v-mos in that they can be rendered tumorigenic by the viral oncogene. However, tumorigenicity was effected through a mechanism which involves different threshold doses for morphologic and tumorigenic transformation.

Further characterization of bleomycin-resistant HeLa cells and analysis of resistance mechanism.

Bleomycin (BLM)-resistant HeLa cells (HeLa-BLMr), which have been subcultured for more than 150 passages during over 2 years in the presence of 1 micrograms/ml of BLM and stably possess a 20-fold-increased BLM-resistance in vitro, were further characterized. The nude mouse tumors produced by HeLa-BLMr were significantly less sensitive (P less than 0.005-0.01) to BLM administration than those produced by HeLa cells, and the cells primarily cultured from nude mouse tumors of HeLa-BLMr and transplanted serially 5 times in the absence of BLM also exhibited a similar degree of BLM resistance to that of HeLa-BLMr cultured in BLM-containing medium. The BLM-resistance mechanism of HeLa-BLMr was partially analyzed. The cells showed about 40% decreased accumulation and 2-3 times reduced retention of [3H]peplomycin, a novel BLM analog, as compared to HeLa cells, but the BLM-hydrolase activity was at almost the same level as that of HeLa cells when determined by HPLC. Furthermore, alkaline sucrose gradient analysis of cellular DNA after BLM treatment revealed that the damaged DNA was more efficiently repaired in HeLa-BLMr than in HeLa cells. These results suggest that decreased drug accumulation and retention, and elevated DNA repair activity are the main mechanism of BLM resistance in HeLa-BLMr.