Tumor-inducing Capacity Research Articles

Abstract 5763: Investigating the carcinogenic potential of various types of mineral fibers in the development of mesothelioma

Abstract Malignant mesothelioma is a very aggressive tumor and has been linked to occupational and environmental exposure to carcinogenic mineral fibers such as asbestos. It was discovered recently that individuals carrying germline BAP1 mutations are predisposed to mesothelioma in presence of even minimal exposure to carcinogenic mineral fibers. Mesothelioma causes about 3,200 deaths per year in the U.S. and over 40,000 deaths per year worldwide. Most malignant mesothelioma studies have focused on carcinogenesis due to occupational exposure to the six regulated industrial mineral fibers collectively called ‘asbestos’. Besides this family of six regulated asbestos minerals, there are other non-regulated fibers that possess physical and chemical structures that are similar to industrial asbestos, and may also be carcinogenic. However, many of those mineral fibers are unknown for their carcinogenesis. We conducted serious studies on this topic and established in vitro and in vivo assays to help determine the carcinogenesis of various types of mineral fibers. We found that high mobility box 1 protein (HMGB1) is a key initiator of asbestos-induced inflammation, which plays critical roles in the development and growth of mesothelioma, and that HMGB1 may be a good indicator for fiber carcinogenesis and a biomarker for fibers exposure. Moreover, we found that specific immune system changes that occur after exposure to carcinogenic fibers, which may be a useful tool to help interrogate the carcinogenic potential of mineral fibers. In particular, the ability to induce an M2 macrophage response appears related to the potent tumor-inducing capacity of carcinogenic mineral fibers. Citation Format: David Larson, Mika Tanji, Andrea Napolitano, Sandra Pastorino, Michele Carbone, Haining Yang. Investigating the carcinogenic potential of various types of mineral fibers in the development of mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5763. doi:10.1158/1538-7445.AM2017-5763

Abstract B23: Epigenetic control of prostate epithelial stem cell differentiation

Abstract Introduction: Hormone manipulation offers an effective but short term and time-limited treatment for human prostate cancers, reducing both tumor bulk and serum levels of prostate-specific antigen. In normal prostate, castration results in a loss of luminal cells and persistence of a poorly differentiated basal-like population, which is responsible for repopulation after restoration of hormone levels. However in fetal prostate development, the hormonal stimulus is provided indirectly to the developing epithelial structures via androgen-sensitive stromal cells. To study the drivers of prostate epithelial cell differentiation, we sought the epigenetic factors affecting cell fate in adult human normal, hyperplastic and malignant epithelium, to provide more precise targets for prostate cancer therapy, based on this developmental paradigm. Methods: We have adopted a multiple model approach for the study of human prostate stem cells. Epithelial cell fractions are prepared from human tissues using multiparametric FACS with a Beckman Coulter Astrios, or adhesion-based separations from (i) MRI guided biopsies of prostate tissues (and patient matched non- tumor tissues) (ii) primary cultures in defined serum free medium from prostate tissues depleted of stromal cells and (iii) a novel set of near patient mouse xenografts of human tissues. In these systems the (normal) colony forming and (malignant) tumor-inducing capacity largely resides in a CD133+/ CD44+/a2B1 integrin Hi/CD49f population which comprises 1% and <0.1% of the normal and malignant epithelium respectively. Cell populations were analyzed independently for their gene expression patterns, non-coding RNA expression, histone modifications and genome CpG methylation. Using a modified analysis algorithm we then related gene expression to both miRNA expression and genomic methylation/ chromatin condensation. Results: At the chromosomal level, prostate SC display a unique pattern of CpG methylation compared to their more differentiated progeny, with distinct changes in cancer. The SC also have a characteristic bivalent chromatin pattern when probed for specific histone modifications. This bivalent chromatin pattern is also indicative, in embryonic systems, of monoallelic gene expression, which was confirmed in prostate SC populations by analysis of the prostate-specific TMPRSS2 gene, in both native and fused form (to ERG). MicroRNA expression patterns are tightly linked to SC differentiation, rather than carcinogenesis, and the miRNAs expressed in prostate SC most closely resemble those in human embryonic SC, and intriguingly in the most malignant forms of prostate cancer. Since miRNAs can control expression of multiple genes, we have devised an algorithm to predict regulation of only those genes expressed in the prostate epithelium. Perhaps significantly for cancer treatment, the principal functions controlled by miRNA expression are both DNA damage repair and wnt signaling, manipulation of which can increase the radiation resistance resident in the SC population. By studying genes which are tightly co-regulated during cell differentiation, we defined 4 non-overlapping sets of genes in which retinoic acid acts as a driver of primitive cell differentiation, an intermediate cell type expressing genes with a bivalent response to RA and androgens, and committed basal/luminal cells in which androgen control of cell fate dominates. Discussion: By studying highly enriched cell populations in different model systems, all of which are derived from primary human tissues, we have built up a complex picture of epigenetic regulation of gene expression during differentiation of human prostate. Many of these essential functions are related to similar functions in developing prostate tissue, but are often characterized by phenotypic plasticity, where the differentiated products of asymmetric division (self-renewal) can revert to a stem-like state. Citation Format: Norman James Maitland, John Packer. Epigenetic control of prostate epithelial stem cell differentiation. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B23.

Comparative study of diagnostic methods used for monitoring of common grape vine (Vitis vinifera L.) crown gall (Agrobacterium vitis Ophel & Kerr) in Slovenia

Agrobacterium vitis causes common grape vine (Vitis vinifera L.) crown gall disease that destroyed a lot of Slovenian vineyards more than a decade ago. Eighty isolates of Agrobacterium spp. collected during monitoring in 2006 were identified as A. vitis and A. tumefacies by pehA and multiplex PCR method. Tumor-inducing capacity of these strains was assessed on test plants and with PCR methods for detection of the Ti plasmid responsible for tumor induction. With VCF3/VCR3 primer pair six false negatives and no false positives were detected. The high genetic diversity of pathogenic Agrobacterium spp. strains affects the performance of molecular methods, thus biological test should be performed where results from molecular methods are doubtful.

Functional analysis of lung tumor suppressor activity at 3p21.3

The early and frequent occurrence of deletions at 3p21.3 in lung cancer has led to the consideration of this chromosomal region as a lung cancer (LUCA) critical region with tumor suppressor activity. We covered this 19 genes-containing region with overlapping P1 artificial chromosomes (PACs), in which genes are likely accompanied by their own promoters or other regulatory sequences. With these PACs we transfected cells from a small cell lung cancer (SCLC) cell line which readily caused tumors in nude mice. Per PAC we selected two cell clones with a low number of PAC copies integrated at a single genomic site. The selected clones were s.c. injected into nude mice to investigate whether the integrated genes suppressed the tumor-inducing capacity of the original SCLC cell line. We could demonstrate PAC-specific gene expression in the transfected cells. All of the PAC integration sites were different. It appeared that introduction of a PAC or even an empty PAC vector causes some chromosomal instability, which in principle may either promote or inhibit cell growth. However, both cell clones with integration of the same PAC from the centromeric part of the LUCA region in different genomic sites were the sole pair of clones that caused smaller tumors than did the original SCLC cell line. This suggests that rather than the induced chromosomal instability, the DNA sequence of that PAC, which in addition to two protein-encoding genes contains at least one potential miRNA gene, is responsible for the tumor suppressor activity.

E1A oncogene induction of cytolytic susceptibility eliminates sarcoma cell tumorigenicity.

The manner in which oncogenes influence tumorigenicity beyond their ability to immortalize cells is uncertain. We tested the hypothesis that, in addition to subverting cellular growth controls, oncogenes can actively determine tumor-inducing capacity by affecting neoplastic cell susceptibility to destruction by the host cellular immune response. The adenovirus type 5 E1A oncogene, which induces susceptibility to lysis by natural killer cells and encodes epitopes recognized by cytotoxic T lymphocytes, was transfected into highly tumorigenic sarcoma cells. E1A expression in these sarcoma cells eliminated their tumorigenicity in recipients with natural killer cell activity that was competent to lyse these E1A-positive targets. Thymus-dependent responses were not required for tumor rejection. These results indicate that oncogene-regulated cellular pathways that affect neoplastic cell susceptibility to natural killer cell lytic mechanisms may influence tumor development in the immunocompetent host.

Intergeneric protoplast fusion between agrobacterium tumefaciens and Bacillus thuringiensis subsp. kurstaki

Intergeneric protoplast fusion betweenA.tumefaciens andB.thuringiensis was performed. The fusants exhibited some properties of both the parental strains. One of the Gram positive fusants with most of theBacillus properties showed tumor inducing capacity in pigeonpea (Cajanascajan).

Viral gene inhibition of class I major histocompatibility antigen expression: not a general mechanism governing the tumorigenicity of adenovirus type 2-, adenovirus type 12-, and simian virus 40-transformed Syrian hamster cells

The association between the level of class I major histocompatibility (MHC) antigen expression and the tumorigenic phenotype was determined for cells from a series of 15 lines of adenovirus type 2 (Ad2)-, Ad12-, and simian virus 40 (SV40)-transformed hamster cells and 16 lines of cells established from hamster tumors induced by SV40 mutants. These cells range from nontumorigenic to highly tumorigenic in both syngeneic and allogeneic adult hamsters. The Ad2-transformed cells--cells that were nontumorigenic in syngeneic adult hamsters--expressed either high levels or low levels of class I MHC antigens. The SV40-transformed cells--cells transformed in vitro that produced tumors with equal efficiency in both syngeneic and allogeneic adult hamsters--or cells derived from SV40-induced tumors expressed very high levels of class I MHC antigens. The Ad12-transformed cells uniformly expressed low levels of class I MHC antigens; these cells produced tumors 200- to 1,000-fold less efficiently in allogeneic adult hamsters than in syngeneic adult hamsters and produced tumors with about the same efficiency in immunoimmature newborns and immunocompetent syngeneic adult hamsters. We conclude that the expression of either high levels or low levels of class I MHC antigens is, at most, a minor factor in the differences observed among these adenovirus- and SV40-transformed cells in their tumor-inducing capacity in naive, immunocompetent hamsters.

Plant-inducible virulence promoter of the Agrobacterium tumefaciens Ti plasmid.

Agrobacterium tumefaciens is the causative agent of crown gall, a plant tumour that can arise on most species of dicotyledonous plants. The tumour-inducing capacity of the bacterium requires the presence of a large plasmid, designated the Ti plasmid, which itself contains two regions essential for tumour formation-the T(umour)-region and the Vir(ulence)-region. The T-region is transferred to plant cells by an unknown mechanism, and becomes stably integrated into the plant genome. The Vir-region has been identified by transposon mutagenesis, but the DNA of this region has never been detected in tumour lines. However, trans-complementation of Vir mutants indicates that genes of the Vir-region are functional in the bacterium. Moreover, the Vir- and T-regions can be physically separated in A. tumefaciens without loss of tumour-inducing capacity. Seven loci, designated virA-F and virO (refs 17, 20-22), have been identified in the Vir-region of the octopine Ti plasmid, but their functions are unknown. As virC mutants in the octopine-type plasmid pTiB6 are invariably avirulent in tests on various plant species, this gene seems to be essential for virulence and we are studying it in detail. We report here that the promoter of virC shows no detectable activity in A. tumefaciens and Escherichia coli K-12 grown in standard medium, but that its activity is induced by a plant product.

Genetic analysis of transfer and stabilization of Agrobacterium DNA in plant cells

In an attempt to elucidate the transfer and integration mechanism of Agrobacterium DNA upon crown gall induction, we translocated a borderless T-DNA to different sites of the C58 Ti plasmid. As a result of the physical linkage of the T-DNA onc genes with other Ti plasmid functions, the concerned strain retained tumor-inducing capacity. However, when the borderless T-DNA is separated on an independent replicon while all other pTi functions are provided in trans, the strain can no longer induce tumors on plants. We provide evidence that the right T-DNA border region harbors one or more in cis active functions essential in the transfer and/or stabilization of the T-DNA into plant cells. The strains used in these experiments allowed us to conclude that some function(s) of the Ti plasmid can induce plant cell proliferations independently of the T-DNA transformation event. The results described here indicate that other Ti plasmid sequences than solely the T-region can be transferred to plant cells.

A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid

The soil bacterium Agrobacterium tumefaciens is a plant pathogen that causes crown-gall tumours after infection of wounded dicotyledonous plants. Large plasmids (Ti-plasmids) are responsible for the oncogenicity of the bacterium1–3. Crown-gall tumours contain a DNA segment, called the T-DNA, which is homologous with a defined part of the Ti-plasmid present in the tumour-inducing bacterium, and is stably integrated into the plant genome4–7. Apart from the T-DNA another region of the Ti-plasmid-called the vir-region, is essential for tumour induction8–11. We report here the interaction of two compatible plasmids, one containing the vir-region, the other carrying the T-DNA on a wide host-range replicon. An A. tumefaciens strain harbouring both plasmids has a normal tumour-inducing capacity, although neither plasmid is functional alone. With this approach, the T-DNA on one plasmid can, because of its size, be easily genetically manipulated using Escherichia coli as a host. Transfer of this plasmid into an A. tumefaciens strain harbouring the plasmid with the vir-region allows introduction of the manipulated T-DNA into plant cells. In this way, sophisticated binary vector systems for plant genetic engineering can be developed.

Presence of allograft-rejection resistance in simian virus 40-transformed hamster cells and its possible role in tumor development.

LSH Syrian hamster cells transformed in vitro by simian virus 40, which is oncogenic for hamsters, are resistant to rejection by adult allogeneic CB hamsters. In contrast, simian virus 40-transformed cells from other species are usually not oncogenic in immunocompetent autologous or isologous hosts. The ability of simian virus 40 to convey resistance to an allograft-type host response to transformed hamster cells may be important in determining the tumor-inducing capacity of these cells and could, in part, explain the species-specific oncogenicity of this virus.

Enhancement of human guanylate cyclase activity by chemical carcinogens.

SummaryThe effect of three carcinogens, streptozotocin, methylnitrosourea, and hydrazine, on guanylate cyclase activity in a variety of human tissues obtained from autopsy specimens 1 to 12 hr after death was examined. Streptozotocin and methylnitrosourea produced two- to sixfold increases in guanylate cyclase activity in lung, liver, colon, kidney, stomach, duodenum, heart, and pancreas. Hydrazine, increased guanylate cyclase activity 21-fold in lung, 52-fold in pancreas, 35-fold in kidney, 24-fold in heart, and 9-fold in liver. The data in this report may help explain the tumor-inducing capacity of these three carcinogens.The authors thank the following for their helpful assistance in obtaining tissue for the study: Drs. Everett V. Sugarbaker, George Irvin, III, Raymond Schaerf, Ronald K. Wright, Elidio C. Fernandez, and Taryn Price. We also thank Dr. Barbara Levey for critical review of this manuscript.These studies were supported in part by NIH Grant No. HL 13715-06, and by a grant from the Juvenile D...

Hydrazine activation of guanylate cyclase: Potential application to tobacco carcinogenesis

Hydrazine, a chemical carcinogen in tobacco and tobacco smoke, has been shown to induce the production of a variety of tumors in rats. The present report demonstrates that hydrazine stimulates the enzyme guanylate cyclase (EC 4.6.1.2) which catalyzes the production of guanosine 3′,5′-monophosphate. At a maximal concentration of 100 mM, hydrazine activated guanylate cyclase 45-fold in liver, 39-fold in colon, 22-fold in stomach, 19-fold in heart, 18-fold in pancreas, 8-fold in lung and kidney, and 4-fold in spleen. Since recent evidence suggests a role for guanosine 3′,5′-monophosphate in malignant transformation, these data may help explain the tumor inducing capacity of this tobacco carcinogen.

Tumour rejection properties of solubilised TSTA from an SV40-induced neoplasm

THE neoplasm mKSA, an SV40-induced sarcoma in BALB/c strain mice has strong, SV40-specific tumour rejection antigens (TSTA). This parental line is also very oncogenic with a tumour dose 50 (TD50) of 102 cells. A subline, passaged in monolayer tissue culture (mKSA-TC), has a much reduced tumour-inducing capacity (TD50 = 2 × 106–2 × 107, cells). Of these mKSA-TC cells 1 × 105 will immunise syngeneic hosts against a challenge of 1 × 106 mKSA cells carried in an ascitic form (mKSA-ASC); immunisation against other SV40-induced BALB/c sarcomas (for example, SVA31C14 and E4) has also been achieved, but not against Adj-PC-5 (ref. 1) nor against Meth A (ref. 2), both neoplasms carrying specific TSTAs and syngeneic with BALB/c but induced by chemical carcinogens.

Acquisition of tumour-inducing ability by non-oncogenic agrobacteria as a result of plasmid transfer.

IT has been suggested that the tumour-inducing capacity of Agrobacterium is determined by plasmid genes1,2. The acquisition of virulence by non-pathogenic agrobacteria from pathogenic agrobacteria has been demonstrated by Kerr3,4. We investigated whether this phenomenon could be the result of transfer of a plasmid. Besides providing further evidence for the hypothesis that plasmid genes carry the genetic information for the tumour-inducing ability of Agrobacterium, the system offers the possibility of studying the genetics and functions of the plasmid.

Further studies on the radioresistance of highly purified mouse and mammary tumor virus

The effects of high doses of γ-rays, ranging from 1080 to 3960 kR, on cell free extracts from mammary tumors and highly purified MTV are compared. Mammary tumor occurrence as well as latency in BALB/c inoculated mice are not affected by the high doses used. Dilutions from 10 −2 to 10 −5 of irradiated samples are not followed by a decrease of biological activity, even at the dilution of 10 −5 which is characterized by a drop in the mammary tumor-inducing capacity of the non-irradiated control specimen. The incubation with ribonuclease crystalline (RNase) ‡ , desoxyribonuclease (DNase) ‡ and pronase B grade (pronase) § § Calbiochem, Los Angeles, California. does not affect the biological activity of irradiated samples. Ultrastructural examination of irradiated virus suspensions shows very rare remnants of MTV compared to the typical aspect of the non-irradiated virus.

Retention of tumour-inducing capacity by adenovirus DNA after cleavage by restriction endonucleases.

WE have previously reported that fragments of Simian adenovirus SA7 DNA produced by physical breakage are oncogenic1. These fragments had an average molecular weight corresponding to half-molecules (11.4× 106). After separation of the heavy and light molecular halves by density gradient centrifugation, we demonstrated that both half-molecule preparations retain the ability to initiate tumours in newborn hamsters. We suggested that this may be caused by the presence of one or more “oncogenic regions” in both molecular halves, or by random breaks near the centre of the DNA which could result in a single small oncogenic region, residing near the centre and occurring with equal probability in either the heavy or light half-molecule preparation. To clarify these results we have studied the effect of several restriction endonucleases on the oncogenic activity of SA7 DNA. These enzymes produce defined fragments of the SA7 genome rather than the heterogeneous populations produced by physical shear.

Anti-tumor Activity in Regression Fluid

Portions of growing tumors were ground in a Waring blender and mixed with Hanks’ balanced salt solution. This tumor homogenate was highly infectious and produced a large proportion of progressive tumors upon inoculation into the wing webs of susceptible birds. However, upon mixing fluid taken from the site of regressing tumors with tumor homogenate and holding it at refrigerator temperature, there was a slight reduction in the ability of the homogenate to produce tumors. A greater reduction of infectivity was obtained when tumor homogenate was mixed with fluid and stored in vivo at the site of regression of a tumor for 24 hours. Mixing regression fluid with Rous sarcoma virus, before inoculation into susceptible White Leghorns, greatly reduced the tumor inducing capacity of the virus. These results suggest the presence of anti-tumor activity in regression fluid. This activity was demonstrated in vitro against Rous sarcoma virus and in vivo against tumor homogenate.