'Vitrified' Dianthus--Teratomata in vitro due to Growth Factor Imbalance

Cultured shoot tips of Dianthus caryophyllus often develop asabnormal, ‘vitrified’ plants that have the characteristicsof teratomata: stable, unlimited development with fasciatedshoot apices, a bush habit, reduced stem elongation and succulentleaves. High concentrations of NAA in the culture medium increasedand benzylaminopurine decreased the proportion of shoot-tipsthat developed as abnormal plants. The requirements of abnormaland normal plants for continued development differed. Duringthe initial stages of development abnormal plants did not requireauxin and they were able to continue their growth in the absenceof roots. It is suggested that the abnormal plants are teratomata,similar to those induced by Crown Gall bacteria, and their inductionand abnormal traits could be due to imbalance of auxins andcytokinins. Dianthus caryophyllus L., carnation, abnormal plant growth, habituation, vitrification, teratoma (plant)     

Regeneration of tobacco plants from crown gall tumors

Summary Tissue culture methods have been developed for regeneration of normal appearing tobacco plants from bacteria-free crown gall strains incited byAgrobacterium tumefaciens C58, IIBV7, B6, CGIC, A6NC, 27, and AT4. Regenerants fall into two categories depending on the properties of tissues from these plants. The first type of regenerant was obtained from tumors incited byA. tumefaciens C58 and it retained the potential for expression of tumor characteristics such as a nonrequirement for phytohormones (auxin and cytokinin) by explants in vitro and the presence of detectable concentrations of nopaline. Normal appearing plants obtained from C58 tumors had much lower concentrations of nopaline than the corresponding tumor tissue (130 versus 1700 μg per g dry wt) indicating a parallel repression of abnormal growth and nopaline concentrations in regenerants. The second type of regenerant was obtained from tumors incited by the otherA. tumefaciens strains and was characterized by requirements for phytohormones by explants in vitro and the apparent lack of octopine or nopaline in regenerant tissues.     

The effect of cytokinins on in vitro cultured inbred lines of Raphanus sativus var. radicula Pers. with genetically determined tumorigenesis

The higher plant tumors are convenient models for studying the genetic control mechanism of plant cell division. There are two types of tumors: induced by the pathogenic factor and genetically determined. The development of both tumor types was related to the changes in cytokinin metabolism and/or signal transduction. In this work, the effect of synthetic cytokinins on the in vitro morphogenesis of cotyledon explants and isolated apices of radish seedlings was studied in several inbred radish lines (Raphanus sativus var. radicula Pers.) that differed in their in vivo tumorigenic properties. It was noted that root formation was stronger affected by kinetin while the treatment with thidiazuron tended to induce active callus formation in cotyledon explants of all inbred lines, except IIa. Growing with benzyladenine produced an intermediate effect as regards all morphogenetic responses. Cytokinin treatment of tumorigenic lines enhanced necrotic development in cotyledon explants. Culturing isolated apices of regenerated plants produced tumors anatomically and morphologically similar to those developing in vivo. Some of the lines nontumorigenic in vivo with enhanced formation of calli on cotyledon explants also developed tumors on apical explants in vitro when treated with cytokinins. These data suggest that different mechanisms for tumor formation operate in various radish lines. The radish lines are classified into three types: (1) necrotic lines with tumor formation putatively related to endogenous cytokinin level, (2) callus-forming lines with cell division enhanced in response to cytokinins, and (3) necrosis-and callus-forming lines with both mechanisms of tumor formation involved.     

Impaired capsule formation of tumors in periostin-null mice

Being a secreted protein, periostin is a multifunctional matricellular glycoprotein. In vitro, periostin has the ability to promote the proliferation and migration of fibroblasts. Previously, it was demonstrated that periostin is mainly produced by cancer-associated fibroblasts or tumor stromal cells. In the present study, we show that periostin regulates capsule formation in a positive manner and inhibits tumor growth. Consistent with a previous finding, several tumor cell lines did not exhibit expression of periostin in vitro or in vivo; and the growth of tumors that had been allografted into periostin −/− mice was significantly accelerated compared with that of the same kind of tumors grafted into periostin +/+ mice. Immunostaining and biochemical analyses revealed that mature collagen was detected abundantly in the capsules and interstitium of the wild-type-grafted tumors but not in those of the periostin −/− grafted tumors. Moreover, the number of activated tumor stromal cells was decreased significantly in the periostin −/− grafted tumors. Our studies suggest that host-derived periostin negatively regulates tumor growth by promoting capsule formation and by mediating changes in the deposition and organization of the tumor microenvironment coordinated by periostin-producing stromal cells.     

Enhancement of Naphthaleneacetic Acid-Induced Rhizogenesis in T(L)-DNA-Transformed Brassica napus without Significant Modification of Auxin Levels and Auxin Sensitivity

Determination of the abscisic acid and indoleacetic acid (IAA) contents of floral stem segments of nontransformed and pRi A₄ T_L-DNA-transformed rape (Brassica napus L. var oleifera, cv Brutor) using a high performance liquid chromatography-enzyme-linked immunosorbent assay procedure and mass spectrometry controls showed that IAA levels were not modified. The regeneration abilities of the in vitro cultured explants were compared on media supplemented with several plant growth regulator combinations. No regeneration occurred on hormone-free media, and shoot production was similar in both genotypes when supplemented with benzyladenine. In the presence of naphthaleneacetic acid (NAA), transformed explants were characterized by faster root regeneration and reduced shoot organogenesis. The optimum for root formation was the same in nontransformed and transformed plants, but the sensitivity threshold was slightly lower in the latter. The NAA inductive period was shorter (14 versus 22 h) with transformed tissue. Root neoformation occurred about 72 h earlier on transformed explants. Our results suggest mainly that there is an acceleration of the auxinic signal transduction and/or that the events preliminary to the formation of roots occur faster in the transformed tissues than in the normal ones.     

Bioassay and attributes of a growth factor associated with crown gall tumors

An improved bioassay is described for a factor that promotes tumor growth which was first obtained from extracts of pinto bean leaves with crown gall tumors. Sixteen primary pinto bean leaves per sample are inoculated with sufficient Agrobacterium tumefaciens to initiate about 5 to 10 tumors per leaf and treated with tumor growth factor at day 3 after inoculation. The diameters of 30 to 48 round tumors (no more than 3 randomly selected per leaf) are measured per test sample at day 6. Mean tumor diameter increased linearly with the logarithm of the concentration of tumor growth factor applied. The tumor growth factor was separated by column chromatography from an ultraviolet light-absorbing compound previously reported to be associated with fractions having maximal tumor growth factor activity. Partly purified tumor growth factor showed no activity in a cytokinin bioassay or an auxin bioassay, and negligible activity in gibberellin bioassays. Representatives of these three classes of growth factors did not promote tumor growth. Extracts from crown gall tumors on primary pinto bean leaves, primary castor bean leaves, Bryophyllum leaves, carrot root slices, and tobacco stems showed tumor growth factor activity, whereas extracts from healthy control tissues did not. Extracts from actively growing parts of healthy pinto beans, Bryophyllum, and tobacco, however, showed tumor growth factor activity. Tumor growth factor is proposed to be a normal plant growth factor associated with rapidly growing tissues. Its synthesis may be activated in nongrowing tissues by infection with Agrobacterium sp.     

Involvement of Tumor Macrophage HIFs in Chemotherapy Effectiveness: Mathematical Modeling of Oxygen,pH, and Glutathione

The four variables, hypoxia, acidity, high glutathione (GSH) concentration and fast reducing rate (redox) are distinct and varied characteristics of solid tumors compared to normal tissue. These parameters are among the most significant factors underlying the metabolism and physiology of solid tumors, regardless of their type or origin. Low oxygen tension contributes to both inhibition of cancer cell proliferation and therapeutic resistance of tumors; low extracellular pH, the reverse of normal cells, mainly enhances tumor invasion; and dysregulated GSH and redox potential within cancer cells favor their proliferation. In fact, cancer cells under these microenvironmental conditions appreciably alter tumor response to cytotoxic anti-cancer treatments. Recent experiments measured the in vivo longitudinal data of these four parameters with tumor development and the corresponding presence and absence of tumor macrophage HIF-1α or HIF-2α in a mouse model of breast cancer. In the current paper, we present a mathematical model-based system of (ordinary and partial) differential equations to monitor tumor growth and susceptibility to standard chemotherapy with oxygen level, pH, and intracellular GSH concentration. We first show that our model simulations agree with the corresponding experiments, and then we use our model to suggest treatments of tumors by altering these four parameters in tumor microenvironment. For example, the model qualitatively predicts that GSH depletion can raise the level of reactive oxygen species (ROS) above a toxic threshold and result in inhibition of tumor growth.     

Hormone-resistant mutants of Arabidopsis have an attenuated response to agrobacterium strains

We have examined the response of the hormone-resistant mutants axr1 and axr2 of Arabidopsis thaliana to inoculation by Agrobacterium tumefaciens and Agrobacterium rhizogenes. Our results indicate that recessive mutations in the axr1 gene affect the frequency of tumor formation after inoculation with either Agrobacterium strain. In addition, tumors produced on axr1 plants were smaller than those growing on wild-type plants. These results indicate that the product of the AXR1 gene is important for both crown gall and hairy root tumor formation. In contrast, the dominant axr2 mutation has a more severe effect on the development of crown gall tumors than on hairy root tumors. Crown gall tumors produced on axr2 plants had a different morphology than wild-type tumors and did not grow when they were removed from the explant. In contrast, a large number of hairy root tumors were produced on wild-type and axr2 plants, and both types of tumors grew when they were removed from the explant. Like the roots of axr2 plants, roots produced on axr2 explants lacked root hairs.     

Hormonal control of tobacco crown gall tumor morphology

The endogenous levels of auxin and cytokinin in teratoma and unorganized tobacco (Nicotiana tabacum L. var Wisconsin #38) crown gall tumor tissues were determined. Teratoma tissues contain levels of auxin and cytokinin favorable for shoot formation, whereas unorganized tumors contain levels of auxin that suppress shoot formation. This conclusion is based upon the observation that when levels of auxin and cytokinin similar to those found in a teratoma were added to the growth medium of nontumorous tobacco tissue, shoot formation resulted; when levels similar to those found in unorganized tumors were added, the normal tissue grew as unorganized callus.     

In VitroModulation of Implantation and Intraepithelial Expansion of Bladder Tumor Cells by Epidermal Growth Factor

A major problem in the management of bladder cancer is the high risk for recurrence of bladder tumors after transurethral resection. This has generally been attributed to the attachment and subsequent expansion of exfoliated tumor cells to the traumatized bladder wall. Anin vitrococultivation model was used to study the implantation and growth of human tumor cells in traumatized murine urothelium. Furthermore, we investigated in a time-course experiment whether stimulation of the regenerative activity of the normal urothelium by a growth factor could affect implantation and subsequent growth of bladder tumor cells. After inoculation on injured confluent cultures of murine urothelium, human T24 and SD bladder carcinoma cells preferentially attached to the denuded areas. SD cells expanded into the normal urothelium as a sharply demarcated tumor, while T24 cells infiltrated as single cells. Treatment of the primary urothelium with epidermal growth factor (EGF) stimulated the proliferation of the primary urothelium and reduced the implantation and growth of T24 considerably. EGF reduced the implantation of the SD tumor cells but could not prevent the further expansion at the expense of surrounding normal urothelium. Since EGF had no effect on migration or proliferation of SD or T24 cells, its modulation of expansive growth is most probably due to an increase in the regeneration of normal urothelium. This study suggests that recurrence of transitional cell carcinomas might in some instances be inhibited by stimulation of the regeneration of traumatized urothelium. The reportedin vitrococultivation model may be useful for studying additional factors involved in intraepithelial expansion of carcinoma cells.     

Inhibition of agrobacterial oncogene expression by means of antisense RNAs

Infection of plants by soil bacterium Agrobacterium tumefaciens induces tumors referred to as crown galls. Tumor development is determined by the introduction of agrobacterial genes governing phytohormone (auxin and cytokinin) production into the plant genome. The most important of these genes are iaaM and ipt. Development of transgenic plants inhibiting the expression of these genes allows a raise of varieties resistant to crown gall disease. For this purpose, single and double tobacco transformants with antisense copies of iaaM and ipt fused with single and double promoters for the 35S RNA of the cauliflower mosaic virus (CaMV 35S and CaMV 35SS) were obtained. Inoculation of transgenic plants harboring the antisense oncogene copies with virulent A. tumefaciens strains C58 (pTiC58) and A6 (pTiA6) revealed significant, but still incomplete, inhibition of these genes. Agrobacterium-mediated transformation of transgenic plants gave rise to weakened tumors, which varied in morphology and allowed regeneration of whole plants. Analysis of the inhibition of the iaaM and ipt expression in tumor cells demonstrated that the RNA interference strategy is promising for developing plant varieties resistant to agrobacterial infection.     

Tumor associated osteoclast-like giant cells promote tumor growth and lymphangiogenesis by secreting vascular endothelial growth factor-C

Tumors with osteoclast-like giant cells (OGCs) have been reported in a variety of organs and exert an invasive and prometastatic phenotype, but the functional role of OGCs in the tumor environment has not been fully clarified. We established tumors containing OGCs to clarify the role of OGCs in tumor phenotype. A mixture of HeLa cells expressing macrophage colony-stimulating factor (M-CSF, HeLa-M) and receptor activator of nuclear factor-κB ligand (RANKL, HeLa-R) effectively supported the differentiation of osteoclast-like cells from bone marrow macrophages in vitro. Moreover, a xenograft study showed OGC formation in a tumor composed of HeLa-M and HeLa-R. Surprisingly, the tumors containing OGCs were significantly larger than the tumors without OGCs, although the growth rates were not different in vitro. Histological analysis showed that lymphangiogenesis and macrophage infiltration in the tumor containing OGCs, but not in other tumors were accelerated. According to quantitative PCR analysis, vascular endothelial growth factor (VEGF)-C mRNA expression increased with differentiation of osteoclast-like cells. To investigate whether VEGF-C expression is responsible for tumor growth and macrophage infiltration, HeLa cells overexpressing VEGF-C (HeLa-VC) were established and transplanted into mice. Tumors composed of HeLa-VC mimicked the phenotype of the tumors containing OGCs. Furthermore, the vascular permeability of tumor microvessels also increased in tumors containing OGCs and to some extent in VEGF-C-expressing tumors. These results suggest that macrophage infiltration and vascular permeability are possible mediators in these tumors. These findings revealed that OGCs in the tumor environment promoted tumor growth and lymphangiogenesis, at least in part, by secreting VEGF-C.     

Functional Expression of TWEAK and the Receptor Fn14 in Human Malignant Ovarian Tumors: Possible Implication for Ovarian Tumor Intervention

The aim of this current study was to investigate the expression of the tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor-inducible 14 (Fn14) in human malignant ovarian tumors, and test TWEAK’s potential role on tumor progression in cell models in-vitro. Using immunohistochemistry (IHC), we found that TWEAK and its receptor Fn14 were expressed in human malignant ovarian tumors, but not in normal ovarian tissues or in borderline/benign epithelial ovarian tumors. High levels of TWEAK expression was detected in the majority of malignant tumors (36 out of 41, 87.80%). Similarly, 35 out of 41 (85.37%) malignant ovarian tumors were Fn14 positive. In these malignant ovarian tumors, however, TWEAK/Fn14 expression was not corrected with patients’ clinical subtype/stages or pathological features. In vitro, we demonstrated that TWEAK only inhibited ovarian cancer HO-8910PM cell proliferation in combination with tumor necrosis factor-α (TNF-α), whereas either TWEAK or TNF-α alone didn’t affect HO-8910PM cell growth. TWEAK promoted TNF-α production in cultured THP-1 macrophages. Meanwhile, conditioned media from TWEAK-activated macrophages inhibited cultured HO-8910PM cell proliferation and invasion. Further, TWEAK increased monocyte chemoattractant protein-1 (MCP-1) production in cultured HO-8910PM cells to possibly recruit macrophages. Our results suggest that TWEAK/Fn14, by activating macrophages, could be ovarian tumor suppressors. The unique expression of TWEAK/Fn14 in malignant tumors indicates that it might be detected as a malignant ovarian tumor marker.     

Studies of thymopoietin pentapeptide (TP5) on experimental tumors: I. TP5 relieves immunosuppression in tumor-bearing mice

The allogeneic and syngeneic immune responses of tumor-bearing mice (C57BL/6 mice bearing 3LL and DBA mice bearing P815) were evaluated by the cytotoxic lymphocyte precursor unit (CLP-U) and MLC. In general, tumor-bearing mice showed slightly enhanced immune responses 4 days after tumor inoculation. This enhanced immune response rapidly declined and about 7–10 days after tumor inoculation, both allogeneic and syngeneic responses were markedly lower than normal. Mice treated with TP5, starting 2 weeks before tumor inoculation, retained normal or enhanced allogeneic and syngeneic responses up to 3 weeks after tumor inoculation. When this tumor-induced suppressive effect was studied in cell transfer experiments, spleen cells from tumor-bearing mice enhanced the growth of tumors in syngeneic recipients whereas spleen cells from TP5-treated mice inhibited the growth of tumors in syngeneic recipients. Moreover, the spleen cells from TP5-treated mice also showed enhanced cytotoxic activity against tumor cells in vitro. These findings suggest that the tumors, after a transient stimulatory phase, induced immune suppressive mechanisms in the hosts' immune defenses. Treatment with TP5 prevented the development of these immune suppressive effects and spleen cells from TP5-treated tumor-bearing mice inhibited tumor growth in freshly tumor-inoculated recipients.     

Chloroplastic and mitochondrial GPX genes play a critical role in rice development

Plant glutathione peroxidases (GPX) catalyze the reduction of H₂O₂ or organic hydroperoxides to water, mitigating the toxicity of these compounds to cells. In rice plants, the GPX gene family is composed of five members that are distributed in a range of sub-cellular compartments including cytosol, mitochondria, chloroplasts, or endoplasmic reticulum. Of these, OsGPX1 and OsGPX4 are located in mitochondria and chloroplasts, respectively. To understand the role of these GPX in rice, the effect of knockdown of OsGPX1 and OsGPX4 in rice plants was evaluated. Our data show that OsGPX4 was essential for in vitro rice regeneration because no plants were obtained from calli carrying a hairpin construct against OsGPX4. Although the knockdown of OsGPX1 did not impair plant regeneration, the plants with silenced OsGPX1 (GPX1s plants) showed reduced shoot length and a reduced number of seeds compared to the non-transformed rice plants. These results indicate that OsGPX1 and OsGPX4 are essential for redox homeostasis which leads to normal growth and development of rice.     

Response of various cucurbits to infection by plasmid-harboring strains of agrobacterium

Tumor formation in cucurbit cultivars resulting from infection by various strains of Agrobacterium tumefaciens and Agrobacterium rhizogenes is environmentally affected. In all instances, tumors could be induced on excised cotyledons while inoculating attached cotyledons or stems resulted in no tumor formation. In addition, buttercup squash (Cucurbita maxima Duch. buttercup) was most susceptible to tumor formation, while butterbush squash (Cucurbita maxima Duch. butterbush) failed to form tumors when inoculated with any of the strains of Agrobacterium. Other tested cucurbit cultivars showed intermediate susceptibility to tumor induction by the various Agrobacterium strains.