Synergistic inhibition of human cancer cell growth by cytotoxic drugs and mixed backbone antisense oligonucleotide targeting protein kinase A

Protein kinase A type I plays a key role in neoplastic transformation, conveying mitogenic signals of different growth factors and oncogenes. Inhibition of protein kinase A type I by antisense oligonucleotides targeting its RIα regulatory subunit results in cancer cell growth inhibition in vitro and in vivo. A novel mixed backbone oligonucleotide HYB 190 and its mismatched control HYB 239 were tested on soft agar growth of several human cancer cell types. HYB 190 demonstrated a dose-dependent inhibition of colony formation in all cell lines whereas the HYB 239 at the same doses caused a modest or no growth inhibition. A noninhibitory dose of each mixed backbone oligonucleotide was used in OVCAR-3 ovarian and GEO colon cancer cells to study whether any cooperative effect may occur between the antisense and a series of cytotoxic drugs acting by different mechanisms. Treatment with HYB 190 resulted in an additive growth inhibitory effect with several cytotoxic drugs when measured by soft agar colony formation. A synergistic growth inhibition, which correlated with increased apoptosis, was observed when HYB 190 was added to cancer cells treated with taxanes, platinum-based compounds, and topoisomerase II selective drugs. This synergistic effect was also observed in breast cancer cells and was obtained with other related drugs such as docetaxel and carboplatin. Combination of HYB 190 and paclitaxel resulted in an accumulation of cells in late S-G₂ phases of cell cycle and marked induction of apoptosis. A cooperative effect of HYB 190 and paclitaxel was also obtained in vivo in nude mice bearing human GEO colon cancer xenografts. These results are the first report of a cooperative growth inhibitory effect obtained in a variety of human cancer cell lines by antisense mixed backbone oligonucleotide targeting protein kinase A type I-mediated mitogenic signals and specific cytotoxic drugs.     

Malignant transformation of Bloom syndrome B-lymphoblastoid cell lines by carcinogens.

Three types of Bloom syndrome B-lymphoblastoid cell lines, as well as one derived from a normal person, treated with 4-nitroquinoline-N-oxide and N-methyl-N'-nitro-N-nitrosoguanidine (0.3 micrograms/ml for 24 hr), were studied for tumorigenicity in nude mice, colony formation in soft agar, cytogenetic changes, and immunoglobulin markers. When normal and Bloom syndrome cells with normal sister chromatid exchange (SCE) levels and karyotypes (type I) were treated with carcinogens, no significant changes occurred in the immunoglobulin profile and karyotype, only rare colony formation was seen, and no tumors were produced. In contrast, when Bloom syndrome cells with high SCE levels (type II with normal karyotype and type III with an abnormal karyotype) were treated with carcinogens, tumors were produced in 22 of 53 nude mice injected; a high rate of colony formation in soft agar was seen; the cells exhibited virtual loss of immunoglobulin markers; and structural changes in chromosomes 1, 2, 3, 4, 5, 7, 11, 14, and 15 were found in the tumors in addition to the original chromosome abnormalities present in the injected cells. It appears that Bloom syndrome B-lymphoblastoid cell lines with high levels of SCE are highly susceptible to the action of carcinogens, as evidenced by tumor formation in nude mice and colony formation in agar. Apparently, the carcinogens were capable of transforming only those cells that had a critical level of SCE (approximately 140 per cell) and not those with only mildly increased levels (approximately 13 per cell).     

KBM-3, an in vitro model of human acute myelomonocytic leukemia.

A human acute myelomonocytic leukemia cell line, KBM-3, was developed to study the pathophysiology of human acute myeloid leukemia. This cell line was characterized by morphology, immunophenotype, Giemsa-banding pattern, in vitro proliferation capacity, and tumorigenicity in nude mice. The KBM-3 cell line was established in the presence of exogenous lymphokines (human placenta-conditioned medium, HPCM), but medium for later passages did not contain HPCM. We found high cellular expression of the mRNA message for granulocyte-macrophage colony-stimulating factor (GM-CSF), which we suggest may be important for the immortalization of the cell line. KBM-3 cells have an immature myelomonocytic phenotype. Cytogenetic analysis revealed a pseudodiploid karyotype with five characteristic marker chromosomes and ranging in total number from 45 to 49. In suspension cultures, the cells had a doubling time of 23 h and a cloning efficiency of about 30% in soft agar independent of exogenous lymphokines. Two-thirds of nude mice injected with 1 x 10(4) KBM-3 cells and all animals injected with 1 x 10(5) cells developed S.C. granulocytic sarcomas within 6-8 weeks. These tumors were locally invasive but did not give rise to distant metastases. When transplanted to a new set of nude mice, all tumors formed secondary sarcomas at the site of implant. We conclude that the KBM-3 cell line may have value for studying the molecular events that underlie the neoplastic transformation in human myeloid leukemia.     

Effects of insulin and somatostatin on the growth and the colony formation of two human pancreatic cancer cell lines

Summary The effects of insulin and somatostatin on the growth and the colony formation of two human pancreatic cancer cell lines, BxPC-3 and SOJ-6, were studied. The BxPC-3 cell line (American Type Culture Collection no. CRL 1687) was derived from a moderately differentiated pancreatic adenocarcinoma. The SOJ-6 cell line is a subclone of SOJ that was initiated from ascites of a well-differentiated pancreatic adenocarcinoma. Both cell lines express fetoacinar pancreatic antigen, an antigen that might be associated with early transformation stages. However, these lines have different proliferation and tumoral powers. SOJ-6 cells showed an almost twofold higher division rate over BxPC-3 cells when cultured in RPMI-1640 medium containing 10% fetal bovine serum. The tumorigenic degree of SOJ-6 cells, as assessed by tumor growth in nude mice, was about three times greater than that of BxPC-3. The in vitro growth of BxPC-3 cells was significantly promoted by insulin, and was slightly inhibited by somatostatin, whereas the growth of SOJ-6 cells was not influenced by these hormones. Using a clonogenic assay in soft agar, the average ratio of colony numbers formed by SOJ-6 and BxPC-3 was about 10/1, indicating a good correlation between the colony formation and tumorigenic degree in vivo. In this test, the number of colonies formed by BxPC-3 cells was increased about twofold in insulin-supplemented medium. On the other hand, somatostatin inhibited the colony formation by a factor of four to six. However, no hormonal modulation of the colony formation of SOJ-6 cells was observed. Our data show that pancreatic cancer cell lines respond differently to pancreatic hormones, and suggest that this may be correlated to a tumour stage or a tumour type.Abbreviations FAP antigen fetoacinar pancreatic antigen - FBS fetal bovine serum - PBS phosphate-buffered saline - RPMI-1640 Roswell Park Memorial Institute's Medium 1640 - CEA carcinoembryonic antigen - CA 19-9 carbohydrate antigen 19-9 This work was financed in part with grant 6394 Association pour la Recherche sur le Cancer (ARC) (Villejuif). Y. Takeda was supported by a fellowship from the ARC     

In vitro growth of colonies of mitogen-stimulated mouse T lymphocytes: I. Conditions affecting colony formation; II. Structure of colonies and component cells

Mouse lymph node cells sensitized with PHA or Con A in liquid phase grew into T-cell colonies when seeded in a two-layer soft agar culture system containing the mitogen. The colony cells were of T-cell lineage. This was deduced from their morphology, ultrastructure, positive strain for theta-isoantigen and the fact that no colonies were formed by lymphoid cells from congenitally athymic nude mice. The architecture of the colonies and their component cells was studied by scanning electron microscopy. Clonogenic assay indicated that macrophages are active modulators of T cell proliferation. Colony formation was markedly enhanced by hemolysate and/or amino acid, L-glutamine or L-cystine, added to the culture medium. The largest number of colonies grew when both the liquid and soft agar media were supplemented with hemolysate and one of the amino acids. Under these conditions the minimal seeding level for colony formation could be reduced from 2.0 X 10(5) to 1.6 X 10(4) cells/culture.     

Interleukin-4 induces a substance in bone marrow stromal cells that reversibly inhibits factor-dependent and factor-independent cell proliferation

Bone marrow-derived stromal cell monolayers pretreated with recombinant interleukin-4 (IL-4) inhibit the growth of hematopoietic cells. This was demonstrated by inhibition of fresh bone marrow-derived, IL-3- induced soft agar colonies as well as by inhibition of proliferation of IL-3-dependent cell lines and of a Friend virus-transformed erythroleukemic cell line. Pretreatment of stromal cells with IL-4 for five to seven days induced the inhibitory activity. IL-4 could then be removed before "plating" the bone marrow cells in soft agar, indicating that the inhibitory activity did not depend on the action of IL-4 on the precursors of the soft agar colonies. The inhibitory activity appears to be mediated by a soluble factor since inhibition was achieved even if the stromal cell layer was separated from the colony forming cells by an "empty" agar layer. However, supernatants of IL-4- induced stromal cell layers had no detectable inhibitory activity. The inhibitory action of the IL-4-pretreated stromal cell lines was not the result of killing of the precursor cells since it could be reversed if the agar layer containing the colony-forming cells was removed from the stromal cell layer and cultured with IL-3. Hydrocortisone (HC) blocked the inhibitory effect if added either in the IL-4 preincubation phase or during the colony formation stage, implying that HC blocked both induction of the inhibitory activity and its release or its effector function. A homogenous long-term stromal cell line could not be induced to exert the inhibitory activity; partial inhibition could be achieved with pure macrophages stimulated with IL-4 and CSF-1, suggesting that the inhibitory activity induced by IL-4 in mixed stromal cell layers may depend on a complex mechanism involving more than one cell type. Northern analysis of RNA from IL-4-induced and uninduced stromal cells indicated that IL-4 did not upregulate expression of CSF-1 or transforming growth factor-beta (TGF-beta) and only modestly increased expression of tumor necrosis factor, suggesting that these cytokines were not responsible for the inhibitory activity. The capacity of IL-4 to induce inhibitory activity in stromal cell layers suggests that IL-4 may play a role in the regulation of hematopoiesis.     

In vitro cell and tissue cultures as a model for studying human tumors

An experimental in vitro or in vivo tumour model should be unchanged represent the biological properties (e.g. histology, proliferation). Changes of tumour cell populations were determined by means of DNA-distribution and multinucleation after cytochalasin B treatment. Flow cytometry measurements on cell cultures in 50 ml glass culture flasks reveal reduction of polyploid cells after collagenase treatment of human mammary carcinomas. Selection of cell populations are responsible for the failed induction of multinucleation by cytochalasin B. In organ cultures the composition of cell population prior to and after 48 hours could maintained. The improved penetration could be demonstrated by autoradiographic measurements of 3H-thymidine incorporation. Thickness, surface and improved penetration of metabolites of vital tissue slices seem to be also important for cell movement and cell division. In 10 out of 12 experiments an earlier cell migration and proliferation could be observed from vital slices than from tissue pieces. Organ cultures represent sufficiently carcinoma in vivo and are more suitable than other mentioned in vitro cell culture methods.     

APO-1基因转染及其抑制大肠癌细胞生长的实验研究

目的 建立表达外源性APO-1基因的大肠癌细胞株,观察APO-1表达细胞株在APO-1抗体作用下对体外培养的大肠癌细胞的抑制效应。方法 采用分子克隆技术将APO-1基因插入真核表达载体pBK-CMV的多克隆位点之间。以脂质体介导法将目的基因导入受体细胞HT-29,用G418筛选克隆细胞。以Northern blot检测转导细胞APO．1mRNA的表达。MTT法和直接记数法以及软琼脂集落形成实验检测转导株在APO-1抗体作用下的细胞增殖水平、生长曲线及细胞克隆形成率。结果 成功地构建了真核表达载体pBK-CMV／APO-1cDNA。转导细胞并经筛选后,获得了2株稳定的抗性细胞,从而建立了APO-1基因表达株(HT-29APO．1cells)。杂交结果表明,转导株APO-1mRNA水平的表达明显高于非转导株。转导细胞增殖速度、倍增时间、对数生长期等均比非转导株更为缓慢和处于抑制状态,集落形成能力低下,但差异无显性意义,而在APO-1抗体作用下效果更为显,差异有非常显性意义。结论 APO-1基因在大肠癌细胞中处于低表达状态;通过真核表达载体的介导,APO-1基因导人大肠癌细胞后,能有效地表达APO-1 mRNA。APO-1表达细胞株在APO-1抗体的作用下可明显抑制体外培养的大肠癌细胞的生长增殖,其作用机制与APO-1诱导细胞凋亡有关。     

CD95抑制大肠癌细胞生长的实验研究

目的建立表达外源性CD95基因的大肠癌细胞株,观察CD95表达细胞株在CD95抗体作用下对体外培养的大肠癌细胞的抑制效应。方法采用分子克隆技术将CD95基因插入真核表达载体pBK-CMV的多克隆位点之间。以脂质体介导法将目的基因导入受体细胞的HT-29,用G418筛选克隆细胞。以Northern blot,Western blot检测转导细胞CD95基因的表达。MTT法和直接记数法以及软琼脂集落形成实验检测转导株在CD95抗体作用下的细胞增殖水平、生长曲线及细胞克隆形成率。结果成功地构建了真核表达载体pBK-CMV/CD95 cDNA。转导细胞并经筛选后,获得了2株稳定的抗性细胞,从而建立了CD95基因表达株(HT-29 CD95 cells)。杂交结果表明、转导株CD95mRNA及其蛋白水平的表达均明显高于非转导株,转导细胞增殖速度、倍增时间、对数生长期等均比非转导株更为缓慢和处于抑制状态,集落形成能力低下,但差异无显著性意义,而在CD95抗体作用下效果更为显著,差异有非常显著性意义。结论 CD95基因在大肠癌细胞中处于低表达状态;通过真核表达载体的介导,CD95基因导入大肠癌细胞后,能有效地表达CD95mRNA及其蛋白。CD95表达细胞株在CD95抗体的作用下可明显抑制体外培养的大肠癌细胞的生长增殖,其作用机制与CD95诱导细胞凋亡有关。     

Effects of okadaic acid on cell growth,anchorage-independent growth,and co-cultures of normal (KMS-6), immortalized (KMST-6), and neoplastically transformed (KMST-6T and KMST-6/RAS) human fibroblasts

The effects of okadaic acid (OA) on normal human (KMS-6), its immortalized (KMST-6) and neoplastically transformed (KMST-6T and KMST-6/RAS) cells were investigated as a model of two-stage carcinogenesis. The presence of OA inhibited cell growth of the normal and immortalized cells but not that of the neoplastic KMST-6T cells. In contrast, cell growth of the other neoplastic KMST-6/RAS cells transformed with the Ha-ras oncogene was inhibited by OA. OA enhanced colony formation of KMST-6T cells in soft agar, but it suppressed that of KMST-6/RAS cells. Co-cultures of KMST-6T cells with normal KMS-6 cells showed an increase in focus formation of KMST-6T cells in the presence of OA, whereas focus formation of KMST-6/RAS cells decreased. These results indicate that OA has growth-promoting effects on certain types of transformed human cells.Abbreviation OA okadaic acid This work was supported by a grant-in-aid from the Ministry of Education, Science, Culture and Sports of Japan     

Loss of growth control and differentiation in the fu-1 variant of the L8 line of rat myoblasts.

A nonfusing variant, fu-1, of the L8 line of rat myoblasts was isolated and characterized with respect to its growth in vitro and developmental properties. Comparative analyses of density-dependent inhibition of growth, serum requirements, cell adhesiveness, colony formation in soft agar, and hexose transport in L8 and fu-1 cells support the conclusion that the fu-1 cells are transformed. In addition, fu-1, but not L8, cells promote the development of tumors in athymic nude mice. fu-1 cells also do not make increased levels of creatinekinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) or myosin and they express an endogenous type-C virus. Both L8 and fu-1 cells express myokinase (ATP:AMP phosphotransferase, EC 2.7.4.3) activities in single cells. In contrast to fu-1 cells, the parent L8 line has increased creatine kinase and myosin after fusion and spontaneously contracts; expression of an endogenous virus could not be detected in these cells. These results suggest that loss of the ability to differentiate normally is associated with the loss of the normal control of cell division of myoblasts grown in vitro and in vivo.     

白藜芦醇对人乳腺癌骨高转移细胞株MDA-MB-231BO生长抑制和凋亡的影响

目的观察白藜芦醇（Res）对人乳腺癌骨高转移细胞株MDA-MB-231BO生长抑制和凋亡的影响,探讨其在抑制乳腺癌骨转移中的应用价值。方法将0、12.5、50、100、200μmo/L的Res分别与MDA-MB-231BO细胞作用24、48、72 h;用CCK-8法检测MDA-MB-231BO细胞的生长抑制率,流式细胞仪分析细胞周期并检测细胞凋亡率,荧光显微镜观察细胞形态。结果随Res浓度的增加及作用时间的延长,MDA-MB-231BO细胞的生长抑制率逐渐升高,S期细胞逐渐增多。Res浓度为200μmol/L、作用48 h,MDA-MB-231BO生长抑制率为82.17%±5.37%、早期凋亡率为4.48%±1.23%、晚期凋亡率为9.48%±2.30%,荧光显微镜下观察用药组随着Res浓度的增加,凋亡细胞逐渐增多。结论Res能抑制人乳腺癌骨高转移细胞株MDA-MB-231BO细胞增殖,使MDA-MB-231BO细胞阻滞于S期,并可诱导该细胞凋亡。     

BRAF V600E maintains proliferation, transformation, and tumorigenicity of BRAF-mutant papillary thyroid cancer cells

CONTEXT: Although the BRAF V600E mutant can initiate the formation of papillary thyroid cancer (PTC), it is unclear whether it is required to maintain cell proliferation, transformation, and tumor growth of BRAF mutation-harboring PTC. OBJECTIVE: The aim of the study was to investigate whether BRAF V600E is required for the proliferation, transformation, and tumorigenicity of BRAF mutation-harboring PTC cells. DESIGN: We addressed this issue using BRAF small interference RNA (siRNA) to transfect stably several BRAF mutation-harboring PTC cell lines, isolated clones with stable suppression of BRAF, and assessed their ability to proliferate, transform, and grow xenograft tumors in nude mice. RESULTS: PTC cell proliferation and transformation were suppressed in specific BRAF siRNA clones, but not in control scrambled siRNA clones. Specifically, taking the advantage of stable BRAF knockdown, we were able to show continued suppression of PTC cell proliferation and transformation, or anchorage-independent colony formation in soft agar, after long-term culture. Moreover, we also demonstrated that in vivo tumorigenicity and growth of tumors from the specific BRAF siRNA cell clones in nude mice were suppressed compared with control clones. CONCLUSIONS: BRAF V600E is not only an initiator of PTC as demonstrated previously but is also a maintainer of proliferation, transformation, and tumorigenicity of PTC cells harboring BRAF mutation, and growth of tumors derived from such cells continues to depend on BRAF V600E. These results provide further support for potentially effective therapy targeted at BRAF for BRAF mutation-harboring PTC.     

Acute myelomonocytic leukemia in children. Possible use of the soft agar culture technique in the differentiation of cellular subtypes.

In 8 children with acute myelomonocytic leukemia (AMML), colony formation in soft agar cultures derived from bone marrow cells was studied in an attempt to differentiate the monocytic (Schilling) from the myelomonocytic (Naegeli) types. The children did not differ markedly in their clinical and morphological parameters. Three in vitro growth patterns were observed: markedly decreased or no growth in 4 cases, extensive growth of granulocytic colonies in 2 cases, and extensive growth of macrophage colonies in the remaining 2. It is suggested that the marrows presenting diminished or no growth patterns are presumably of acute myelogenous leukemia patients with a monocytic component. The excessive granulocytic or macrophage colony growth may be an in vitro indication for an in vivo proliferation of either granulocytic or monocytic leukemic cell lines, and therefore may represent the Naegeli or Schilling variants of AMML respectively. If these observations can be approved in a larger series of AMML patients, this approach can be valuable as another tool in the differential diagnosis of the subtypes of AMML in children.     

Differential effects of red cells on the formation of normal and neoplastic mouse B lymphocyte colonies in vitro.

Intact sheep red cells potentiated mouse B lymphocyte colony growth in agar but red cell membranes or lysates exhibited no comparable ability. For maximum colony formation red cells had to be present throughout the entire culture period. Red cells added to cultures late in the culture period did not potentiate growth. Intimate contact between colony-forming cells and red cells was not essential for potentiation to occur. Red cell lysates inhibited normal B lymphocyte colony formation in cultures containing intact red cells, but did not inhibit colony formation by cells of the B lymphoid leukemia ABE-8. This differential effect may provide a means of differentiating normal from neoplastic colony-forming B lymphocytes in the mouse. Eight different tumors were also examined. Intact red cells potentiated colony formation by all of them. Lysed red cells did not potentiate the growth of any of the tumor lines. The mastocytoma P815 was the only tumor whose colony formation was inhibited by the addition of intact and lysed red cells.     

Neoplastic transformation of chimpanzee cells induced by adenovirus type 12--simian virus 40 hybrid virus.

The adenovirus 12--simian virus 40 hybrid virus produced neoplastic transformation of chimpanzee skin fibroblasts in vitro. The transformed fibroblasts showed morphological alteration and became permanent lines. The transformed cells contained both adenovirus 12 and simian virus 40 large tumor antigens and were virus producers. However at passage 9, one line (WES) was found to be a nonproducer, producing neither infectious virus nor virus-specific antigen detectable by the complement fixation test. Virus particles were not detected nor could infectious hybrid virus be rescued from this line by cocultivation with Vero cells. The transformed cells formed large cell aggregates and grew in liquid growth medium above an agar base, formed colonies in soft agar, and grew to high saturation densities; the normal chimpanzee skin fibroblasts did not. One transformed WES line produced tumors when transplanted subcutaneously into newborn nude mice, thus providing an important tool for studying tumor immunity in the chimpanzee.     

RNAi抑制存活素基因表达对前列腺癌细胞侵袭的影响

目的 探讨存活素(survivin)基因对前列腺癌细胞侵袭的影响和可能机制. 方法 采用survivin基因小干扰RNA(small interfering RNA,siRNA)转染处理人前列腺癌细胞系PC-3后,分别采用实时-定量RF PCR和Western blot检测survivin基因和基质金属蛋白酶家族-2、-9(matrixmetalloproteinases-2、-9)mRNA和蛋白水平,分别采用软琼脂集落培养试验和Boyden小室模型试验检测癌细胞的锚着不依赖性增殖和侵袭能力;其次,将转染48 h的细胞接种裸鼠,观察对癌细胞体内侵袭的影响. 结果 软琼脂集落形成试验显示,3.125、6.250 nmol/L和12.500 nmol/L siRNA组集落形成数分别为17.8±1.6、13.6±1.5、8.8±1.4,而对照组为22.65±1.8(P<0.05);Boyden小室模型试验显示,3.125、6.250 nmol/L和12.500/ nmol/L siRNA组穿过滤膜的细胞数分别为33.6±2.1、19.5±1.9、8.1±1.8,而对照组为49.4±2.3(P<0.05).体内试验显示,对照组裸鼠组织癌细胞侵袭横纹肌和血管,而转染组未见这些现象.同时发现,转染组细胞MMP-2、-9基因mRNA和蛋白水平明显下调,呈浓度和时间依赖性(P<0.01). 结论 采用survivin siRNA转染可抑制前列腺癌细胞侵袭转移,其机制可能与下调MMP-2和MMP-9表达有关.     

Correlation of patterns of anchorage-independent growth with in vivo behavior of cells from a murine fibrosarcoma.

The pattern of in vitro anchorage-independent growth of tumor cells from the murine UV-2237 fibrosarcoma correlated with their ability to produce experimental metastasis in vivo. When seeded into 0.3% Noble agar semisolid medium, cells of metastatic clones developed into larger tumor colonies at a faster rate than did cells of clones with low metastatic potential. Furthermore, when tumor cells were plated into 0.6% Noble agar, colony development by cells of low metastatic potential clones was almost completely restricted. Tumor cells from the heterogeneous parent UV-2237 fibrosarcoma were plated into dishes containing 0.6% agar semisolid medium. In separate experiments, 16 colonies were isolated 2 weeks thereafter and were established as individual cell lines in monolayer cultures. All of these cell lines produced experimental metastases as determined by in vivo lung colony assay. The data suggest that anchorage-independent growth of UV-2237 tumor cells in 0.6% Noble agar semisolid medium is selective and permits the isolation of metastatic subpopulations.