Characterization of human mammary normal and neoplastic cells grown in culture

The paper presented here describes cultivation and characterization of human mammary normal (NMC) and neoplastic (BO) cells. Characteristics: growth rate, colony growth in soft agar, nuclear overlaps, induction of multinucleation by Cytochalasin B and transplantation in vivo were compared between NMC- and BO-cells. Normal (NMC) cells are characterized by a slow growth rate (cell doubling time less than 70 hours), no growth in soft agar, no induction of uncontrolled nuclear division and no development of tumors after transplantation of cells in nude mice in vivo. The two cell types do not differ with respect to the nuclear overlapping ratio. In contrast, neoplastic (BO) cells showed a lower doubling time (less than 20 hours), colony growth in soft agar (greater than 20 colonies/30,000 cells), induction of multinucleation by Cytochalasin B (greater than or equal to 3 nuclei/cell), and tumors in nude mice were observed. Both cell types were recommended as an in-vitro model for screening antineoplastic agents.     

Induction of apoptosis by wild-type p53 in a human colon tumor-derived cell line.

A wild-type p53 gene under control of the metallothionein MT-1 promoter was stably transfected into human colon tumor-derived cell line EB. Repeated inductions of the metallothionein wild-type p53 gene with zinc chloride results in progressive detachment of wild-type p53 cells grown on culture dishes. Examination at both the light and electron microscopic level revealed that cells expressing wild-type p53 developed morphological features of apoptosis. DNA from both attached and detached cells was degraded into a ladder of nucleosomal-sized fragments. Expression of wild-type p53 inhibited colony formation in soft agar and tumor formation in nude mice. Furthermore, established tumors in nude mice underwent regression if wild-type p53 expression was subsequently induced. Regressing tumors showed histological features of apoptosis. Thus, regression of these tumors was the result of apoptosis occurring in vivo. Apoptosis may be a normal part of the terminal differentiation program of colonic epithelial cells. Our results suggest that wild-type p53 could play a critical role in this process.     

Analyses of bromodeoxyuridine-associated sister chromatid exchanges (SCEs) in Bloom syndrome based on cell fusion: single and twin SCEs in endoreduplication.

When Bloom syndrome (BS) cells labeled with bromodeoxyuridine (BrdUrd) for one round of DNA replication were fused with nonlabeled normal cells, the hybrid cells had a normal level of sister chromatid exchange (SCE) at the first mitosis after fusion. However, when normal cells treated with mitomycin C (MC) were fused with nontreated normal cells, the MC-induced SCE was not affected by fusion with normal cells. Single and twin SCEs were analyzed in the Colcemid-induced endoreduplicated normal and BS lymphoid B cells from diplochromosomes. In normal cells, the same number of SCEs occurs in each of the two cell cycles; the SCE ratio of single (6.30 SCEs per cell) to twin (2.92 SCEs per cell) was 2:1 on the endoreduplicated-cell basis, showing 1:1 on the diploid-cell basis. In BS cells, the SCE ratio of single (144.8 SCEs per cell) to twin (5.9 SCEs per cell) was 25:1 on the endoreduplicated-cell basis and was 12:1 on the diploid-cell basis. These studies strongly suggest that most of the BS SCEs occur during the second cell cycle when BrdUrd-containing DNA is used as template for replication and that the normal level of BS SCE observed at the first mitosis of the hybrid cells is the result of SCE inhibition resulting from the fusion with normal cells.     

Requirement of phospholipase D1 activity in H-RasV12-induced transformation

The ability of the Ras oncogene to transform normal cells has been well established. One downstream effector of Ras is the lipid hydrolyzing enzyme phospholipase D. Recent evidence has emerged indicating a role for phospholipase D in cell proliferation, membrane trafficking, and migration. To study the potential importance of phospholipase D in the oncogenic ability of Ras, we used Rat-2 fibroblasts with reduced phospholipase D1 activity (Rat-2V25). Here, we show that H-Ras transformation of Rat-2 fibroblasts requires normal phospholipase D1 activity. WT Rat-2 fibroblasts transfected with the H-RasV12 oncogene grew colonies in soft agar and tumors in nude mice. However, Rat-2V25 cells when transfected with the H-RasV12 oncogene did not form colonies in soft agar or produce tumors when xenografted onto nude mice. Interestingly, in the presence of phosphatidic acid, the product of phospholipase D, growth in soft agar and tumor formation was restored. We also observed a dramatic increase in the expression of phospholipase D1 in colorectal tumors when compared with adjacent normal mucosa. Our studies identify phospholipase D1 as a critical downstream mediator of H-Ras-induced tumor formation.     

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.     

Corrective factor of bloom syndrome: Further characteristics

Bloom syndrome is an autosomal recessive inherited disease characterizedin vitro by chromosomal instability and an increased rate of sister chromatid exchanges (SCE). Co-cultivation of Bloom syndrome and normal fibroblasts results in correction of chromosomal instability in Bloom syndrome. We report our studies of the effects of co-cultivation on homozygous Bloom cells with heterozygous cells. Additionally, we used fibroblasts from a patient with Fanconi anaemia and with xeroderma pigmentosum.     

Growth of cultured cells from patients with Hodgkin''s disease and transplantation into nude mice.

Long-term monolayer cell cultures have been prepared from tumor nodules in spleens removed from 28 patients with Hodgkin's disease and from 84 spleens that did not have tumors from Hodgkin's disease patients, normal adult spleens, and human fetal spleens and thymuses. After 5 to 20 serial passages in culture, cells from nine of the Hodgkin's disease monolayers underwent morphologic change in vitro with transition from a spindle and reticular pattern of replication to polygonal and round cells that propagated in mosaic arrays. Four of such Hodgkin's disease monolayer cell lines were injected subcutaneously into 43 nude, athymic mice. In 36 animals (84%), neoplasms developed at the inoculation site that werel ocally destructive, capable of pulmonary metastasis, and eventually fatal to the recipients. Transplanted tumors were not observed in 18 athymic mice injected with cultures prepared from normal human adult spleen and fetal spleen and thymus, nor were tumors seen in 16 similar animals that received fresh, noncultured Hodgkin's disease tumor tissue. On microscopic examination, xenografts derived from Hodgkin's disease cultures were pleomorphic malignant neoplasms composed of large, undifferentiated cells, resembling reticulum cell sarcoma. These neoplasms did not involve the lymphoreticular organs of mice. Chromosome studies indicated that the transplanted neoplasms were composed of human cells with an aneuploid karyotype and that monolayer cultures prepared from the heterotransplants contained a karyotype similar to that of the cultured cells prior to passage in mice. The ability of these Hodgkin's disease cell lines to produce invasive tumors with human karyotypes in nude mice is evidence of the neoplastic nature of the monolayer cells and their relationship to the malignant cell of the human disorder.     

Proliferation of human malignant hematopoietic cells in immunodeficient mice: suppression by antibody to pluripotent K-562 leukemia cells involves direct cytolysis and effector cells

Six human hematopoetic cell lines were successfully heterotransplanted into athymic (nude) and asplenic-athymic (lasat) neonatal mice. The tumors arising from leukemia and lymphoma cells could then be serially transplanted into adult nude mice. Seven days after the fourth serial mouse passage, each mouse was treated with goat immune gamma globulin against K-562 cells. One control group was treated similarly, but with nonimmune (normal) gamma globulin, while another control group was not treated. The goat gamma globulin was not toxic for nude and lasat mice, and the immune, but not the normal, gamma globulin suppressed local subcutaneous growth of myelosarcomas, lymphosarcomas, and Burkitt lymphoma cells. On the other hand, the growth of lung, breast, and prostatic carcinomas and a melanoma of human origin were not altered by the immune gamma globulin. Since suppression of cell growth occurred equally well in decomplemented mice, a complement-mediated cytotoxicity apparently cannot be considered as responsible for the abrogation. The Fab fragment of the immunoglobulin did not suppress the growth of the myelosarcomas. We conclude that antibody suppression of the in vivo proliferation was specific for malignant hematopoietic cells and that the Fc portion of IgG is necessary for in vivo cytolysis of leukemia cells. The most probable mechanisms are direct antibody cytolysis and antibody-dependent macrophage-mediated cytotoxicity.     

Anchorage-independent growth of normal human fibroblasts.

Normal human fibroblasts, considered to be entirely anchorage dependent for proliferation, have been grown in methylcellulose medium. The most important factor required for growth in suspension appears to be the use of high levels of serum and hydrocortisone. Newborn foreskin or fetal lung fibroblasts form colonies as large as 0.5 mm in diameter after 3 wk, with a colony-forming efficiency as high as 70%. Mouse 3T3 cells that do not form colonies in standard assays for anchorage-independent growth also grow under these conditions. Colony formation results after inoculation of as few as 100 cells per 60-mm dish, and metaphase cells have been visualized with a fluorescent DNA stain, showing that colony formation is due to division rather than aggregation. Fibroblasts recovered from suspension and grown as monolayers retain a diploid karyotype and normal shape, do not form tumors upon injection into nude mice, and become senescent. Thus, the trait of anchorage-independent growth in vitro is clearly possessed by normal human fibroblasts and can be expressed under the proper conditions.     

Skp2 is oncogenic and overexpressed in human cancers

Skp2 is a member of the F-box family of substrate-recognition subunits of SCF ubiquitin-protein ligase complexes that has been implicated in the ubiquitin-mediated degradation of several key regulators of mammalian G(1) progression, including the cyclin-dependent kinase inhibitor p27, a dosage-dependent tumor suppressor protein. In this study, we examined Skp2 and p27 protein expression by immunohistochemistry in normal oral epithelium and in different stages of malignant oral cancer progression, including dysplasia and oral squamous cell carcinoma. We found that increased levels of Skp2 protein are associated with reduced p27 in a subset of oral epithelial dysplasias and carcinomas compared with normal epithelial controls. Tumors with high Skp2 (>20% positive cells) expression invariably showed reduced or absent p27 and tumors with high p27 (>20% positive cells) expression rarely showed Skp2 positivity. Increased Skp2 protein levels were not always correlated with increased cell proliferation (assayed by Ki-67 staining), suggesting that alterations of Skp2 may contribute to the malignant phenotype without affecting proliferation. Skp2 protein overexpression may lead to accelerated p27 proteolysis and contribute to malignant progression from dysplasia to oral epithelial carcinoma. Moreover, we also demonstrate that Skp2 has oncogenic potential by showing that Skp2 cooperates with H-Ras(G12V) to malignantly transform primary rodent fibroblasts as scored by colony formation in soft agar and tumor formation in nude mice. The observations that Skp2 can mediate transformation and is up-regulated during oral epithelial carcinogenesis support a role for Skp2 as a protooncogene in human tumors.     

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.     

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.     

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.     

Establishment and characterization of a new cell line (VHB-1) derived from a primary breast carcinoma

Summary A continous line of human breast carcinoma cells, VHB-1, was established in culture following collagenase treatment of an infiltrating duct cell carcinoma. The cells displayed an epithelial pattern and multiplied rapidly. Maintained in monolayer culture, the VHB-1 cells exhibited a 30-h doubling time and a plating efficiency of 20%. The cells possessed an abnormal karyotype with a mode of 70–74 chromosomes per cell. the karyotype was heavily rearranged and numerous marker chromosomes were found. Transplantation of the cells into nude mice produced tumors bearing histological resemblance to the original material. The VHB-1 cells contained significant levels of prolactin receptors, were steroid hormone (estrogen, progesterone, androgen, glucocorticoid) receptor positive, and were capable of functional differentiation in vitro. These characteristics make the VHB-1 cell line a suitable model for studying the biological properties of human breast tumors.     

Heterogeneity of cancer cells from a single human colon carcinoma

The human colon carcinoma cell line DLD-1, established from tumor tissue obtained from a 45 year old white man with an adenocarcinoma of the sigmoid colon, was studied from the perspective of tumor heterogeneity. The karyotype and morphology of cells from an early passage DLD-1 culture, as well as the histologic features of both the original tumor and neoplasms produced by inoculation of athymic nude mice with DLD-1 cells, indicated that both the DLD-1 cell line and the original tumor were heterogeneous. Two clones were isolated from the DLD-1 line; they differed in their morphology, karyotype, and cloning efficiency in soft agar. Furthermore, when cells from each clone were injected into athymic mice, histologically distinct tumors were produced. Various analyses showed that the two cloned lines were representative of the two subpopulations predominantly responsible for the heterogeneity of the original neoplasm. In vitro drug screening results demonstrated that the two cloned lines have differential sensitivities to chemotherapeutic agents. The parent DLD-1 human colon carcinoma cell line and its two cloned subpopulations provide material for the study of various aspects and implications of human cancer cell heterogeneity.     

T cells induce terminal differentiation of transformed B cells to mature plasma cell tumors.

Major interest in the analysis of mature plasma cell neoplasias of mice and humans has focused on identification of precursor cells that give rise to mature malignant plasma cells. Although several laboratories have recently suggested that such cells are present in the granulomas of pristane-treated mice and the bone marrow of some multiple myeloma patients, the in vivo cellular interactions required for their differentiation into mature plasma cell tumors remains unclear. Given the extensive interactions of peripheral T cells and normal B cells, we assessed the potential role of T cells in plasma-cell tumor development, by using a myc, raf-containing retrovirus, J3V1, to induce plasmacytomas in normal BALB/c mice, T-cell-deficient nude mice, and T-cell-reconstituted nude mice. The B-lineage tumors arising in normal BALB/c mice were uniformly mature plasmacytomas, most of which secreted immunoglobulin. In contrast, nude mice yielded predominantly non-immunoglobulin-secreting B-cell lymphomas with a phenotype characteristic of peripheral B cells. T-cell reconstitution of nude mice prior to tumor induction resulted in a shift from B-cell lymphomas to plasmacytomas. These results imply that transformation can occur prior to terminal differentiation of B cells and that such transformed cells can be driven to terminal differentiation by peripheral T cells. These findings further suggest that, in human multiple myeloma, the ability of T cells to influence the differentiation state of transformed B cells may provide a mechanism by which malignant plasma cells found in the bone marrow could arise from clonotypically related less-mature B cells found in both the bone marrow and periphery.     

The effect of syngeneic peripheral blood cells on the formation of colonies by normal human bone marrow cells in diffusion chambers in mice.

This paper describes the influence of cells capable of releasing colony stimulating activity (CSA) in vitro on the formation of granulocytic colonies by normal human bone marrow in diffusion chambers in mice. A carbonyl iron method was used to remove phagocytic cells from normal human bone marrow. This treatment prevented spontaneous colony and cluster formation when the cells were cultured in agar in vitro at initial concentrations of 2-5 x 105 cells per ml. However, non-phagocytic bone marrow cells formed granulocytic colonies when inoculated into diffusion chambers at 105 cells per chamber and cultured in 450 R-irradiated or non-irradiated mice. The formation of granulocytic colonies by carbonyl iron treated marrow in diffusion chamber cultures was not consistently enhanced by the admixture of 1.4 x 105 1500 R-irradiated syngeneic light density blood cells (less than 1.077 g/ml) to the chamber inoculum. In contrast, these cells induced cluster or colony formation when added in the same proportion to the marrow cells in agar cultures in vitro. Addition of 1.4 x 105 1500 R-irradiated high density blood cells(greater than 1.077 g/ml) to the inoculum resulted in a slight, non-significant decrease in the number of colonies in diffusion chambers. The stimulating effect of host irradiation on neutrophilic colony formation was independent of the presence of CSA releasing cells in the chamber inoculum.     

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.     

Studies on the pathogenesis of refractory anemia

Nine patients with refractory anemia were studied using the soft agar marrow culture assay (CFU-c) to identify granulocyte-monocyte progenitor cells. Patients' marrows were then cocultured with normal marrow to identify suppressor cells that inhibit normal colony formation. Three of nine patients had low colony formation and no suppression in coculture. These patients may have a defect intrinsic to the marrow granulocyte-monocyte progenitor cell, termed type I. Three of nine patients had normal colony formation and no suppression in coculture, possibly representing a type II defect in the hemopoietic environment. Three of nine patients had low colony formation in the CFU-c assay and their marrow contained cells that suppressed colony formation by normal marrow in coculture. This defect, termed type III, may result from suppressor cells. Thus, refractory anemia may be a syndrome resulting from at least three different pathogenetic mechanisms involving defects in (1) stem cells, (2) the marrow environment or (3) suppressor cells. This may represent one end of the spectrum of pancytopenia with diminished cellularity (aplastic anemia) or normal cellularity (refractory anemia) resulting from similar mechanisms.     

Assessment of pancreatic islet-cell population in the hyperglycemic athymic nude mouse: immunohistochemical, ultrastructural, and hormonal studies

Previous studies in diabetic animal models have demonstrated altered pancreatic islet-cell populations. To further characterize the diabetic syndrome in our athymic nude mouse colony, we studied the population of endocrine cells in pancreatic islets of 4-week-old normoglycemic and 8-week-old hyperglycemic athymic nude (nu/nu) mice using immunohistochemistry, morphometry, and electron microscopy. In normoglycemic 4-week athymic nu/nu mice, the proportions of B (insulin-secreting) cells and A (glucagon-secreting) cells were similar to those in control Balb/c mice; however, the D (somatostatin-secreting) cells were significantly decreased in nu/nu mice. The populations of B and A cells appeared to be normal in hyperglycemic 8-week-old nu/nu mice while there was a significant increase in the proportion of D cells when compared with the proportion in Balb/c mice. Electron microscopic studies indicated that the appearance of B and A cells was similar in the 8-week-old hyperglycemic nu/nu and in controls; however, the D cells appeared to be enlarged and were finely packed with electron-dense secretory granules. Radioimmunoassays of the pancreatic content (micrograms/g fresh pancreas) of insulin, glucagon, and somatostatin in pancreata in 8-week-old normal Balb/c and hyperglycemic athymic nude mice were similar; however, the somatostatin content was significantly increased in the 8-week-old hyperglycemic nu/nu mice compared with age and sex-matched controls. These results demonstrate an altered D cell population and an increase in somatostatin levels in the pancreatic islets of the hyperglycemic athymic nude mouse animal model.(ABSTRACT TRUNCATED AT 250 WORDS)