Cytomorphological lesions induced by chemotherapeutic agents in transitional cell carcinoma in vitro

Summary Transitional cell carcinoma from 20 patients and two human cell lines were maintained in short-term tissue culture. Each was studied ultrastructurally before and after incubaton with cisplatinum, adriamycin, or mitomycin C. Sequential ultrastructural changes were noted and were found to be specific for each agent tested. Ultrastructural changes in the nucleoli were produced by exposure to cisplatinum or mitomycin C; alterations in the heterochromatin of the nuclei were characteristic of treatment with adriamycin. The changes in the nucleoli seen with cisplatinum have not been described previously and support an alkylating property as a mechanism of action. Intravesical chemotherapeutic agents are now commonly used in clinical treatments. The morphological changes produced by these agents are specific and may be seen in the clinical setting. This research was supported by the Veteran's Administration Merit Review grant.     

Cell volume growth after cell cycle block with chemotherapeutic agents.

The effects of various chemotherapeutic agents on the volume of Chinese hamster V79 fibroblasts and murine lymphoma L5178Y cells were studied by electronic volume spectroscopy. Cells arrested in the division cycle by a chemotherapeutic block continued to grow in volume resulting in abnormally large cells unable to reduce their volume by cell division. This was observed in cells treated with colcemid, vinblastine, excess thymidine, hydroxyurea, ARA-C, 5-fluorouracil, actinomycin-D and bleomycin, but not with puromycin or cycloheximide. Increase in cell volume of blocked cells was correlated with a decrease in cell survival as measured by clonogenic ability. The results suggest the possibility of volume spectroscopy for a rapid in vitro test to determine tumor sensitivity to chemotherapeutic agents and the in vivo monitoring of response to chemotherapy. Mechanisms for increased cell kill by a second agent acting selectively on enlarged cells are considered.     

Epithelial cell spreading induced by hepatocyte growth factor influences paxillin protein synthesis and posttranslational modification

Superficial wounds in the gastrointestinal tract rapidly reseal by coordinated epithelial cell migration facilitated by cytokines such as hepatocyte growth factor (HGF)/scatter factor released in the wound vicinity. However, the mechanisms by which HGF promotes physiological and pathophysiologic epithelial migration are incompletely understood. Using in vitro models of polarized T84 and Caco-2 intestinal epithelia, we report that HGF promoted epithelial spreading and RhoA GTPase activation in a time-dependent manner. Inducible expression of enhanced green fluorescent protein-tagged dominant-negative RhoA significantly attenuated HGF-induced spreading. HGF expanded a zone of partially flattened cells behind the wound edge containing basal F-actin fibers aligned in the direction of spreading. Concomitantly, plaques positive for the focal adhesion protein paxillin were enhanced. HGF induced an increase in the translation of paxillin and, to a lesser extent, beta1-integrin. This was independent of cell-matrix adhesion through beta1-integrin. Subcellular fractionation revealed increased cosedimentation of paxillin with plasma membrane-containing fractions following HGF stimulation, without corresponding enhancements in paxillin coassociation with beta1 integrin or actin. Tyrosine phosphorylation of paxillin was reduced by HGF and was sensitive to the Src kinase inhibitor PP2. With these taken together, we propose that HGF upregulates a free cytosolic pool of paxillin that is unaffiliated with either the cytoskeleton or focal cell-matrix contacts. Thus early spreading responses to HGF may partly relate to increased paxillin availability for incorporation into, and turnover within, dynamic cytoskeletal/membrane complexes whose rapid and transient adhesion to the matrix drives migration.     

Akt promotes increased mammalian cell size by stimulating protein synthesis and inhibiting protein degradation

Expression of constitutively active Akt3 was found to increase the size of MCF-7 cells approximately twofold both in vitro and in vivo. A regulatable version of Akt1 (MER-Akt) was also found capable of inducing a twofold increase in the size of H4IIE rat hepatoma cells. Rapamycin, a specific inhibitor of mTOR function, was found to inhibit the Akt-induced increase in cell size by 70%, presumably via inhibition of the Akt-induced increase in protein synthesis. To determine whether Akt could be inhibiting protein degradation, thereby contributing to its ability to induce an increase in cell size, we conducted protein degradation experiments in the H4IIE cell line. Activation of MER-Akt was found to inhibit protein degradation to a degree comparable to insulin treatment. The effects of these two agents on protein degradation were not additive, thereby suggesting that they were acting on a similar pathway. An inhibitor of the phosphatidylinositol 3-kinase pathway, LY-294002, blocked both insulin- and Akt-induced inhibition of protein degradation, again consistent with the hypothesis that both agents were acting on the same pathway. In contrast, rapamycin did not block the ability of either agent to inhibit protein degradation. These results indicate that Akt increases cell size through both mTOR-dependent and -independent pathways and that the latter involves inhibition of protein degradation. These studies are also consistent with the hypothesis that insulin's ability to regulate protein degradation is to a large extent mediated via Akt.     

Inflating bacterial cells by increased protein synthesis

Understanding how the homeostasis of cellular size and composition is accomplished by different organisms is an outstanding challenge in biology. For exponentially growing Escherichia coli cells, it is long known that the size of cells exhibits a strong positive relation with their growth rates in different nutrient conditions. Here, we characterized cell sizes in a set of orthogonal growth limitations. We report that cell size and mass exhibit positive or negative dependences with growth rate depending on the growth limitation applied. In particular, synthesizing large amounts of “useless” proteins led to an inversion of the canonical, positive relation, with slow growing cells enlarged 7‐ to 8‐fold compared to cells growing at similar rates under nutrient limitation. Strikingly, this increase in cell size was accompanied by a 3‐ to 4‐fold increase in cellular DNA content at slow growth, reaching up to an amount equivalent to ~8 chromosomes per cell. Despite drastic changes in cell mass and macromolecular composition, cellular dry mass density remained constant. Our findings reveal an important role of protein synthesis in cell division control.     

Comparison of protein synthesis inhibition kinetics and cell killing induced by immunotoxins

Immunotoxins comprised of a monoclonal antibody covalently coupled to recombinant ricin A chain or to a binding-defective form of diphtheria toxin were compared with respect to their rates of protein synthesis inhibition and efficiencies of killing target cells. Protein synthesis inhibition rates were established by measuring the incorporation of L-[14C]leucine in toxin-treated cells relative to untreated cells at several times after exposure of cells to an immunotoxin. Cell killing was assessed by a limiting dilution assay which measures the number of cells surviving toxin treatment relative to untreated cells. At equivalent protein concentrations, the diphtheria toxin immunotoxin inhibited protein synthesis significantly more rapidly than the ricin A immunotoxin but, contrary to previous predictions, achieved a significantly lower cell kill. Thus, the kinetics of protein synthesis inactivation do not necessarily correlate with killing efficiencies. Possible explanations for these results are that the effect of the diphtheria toxin immunotoxin on protein synthesis is partially reversible or that the diphtheria toxin immunotoxin enters the cytosol at a faster rate than the ricin A immunotoxin but also is degraded at a faster rate.     

Prevention by the MER tubercle bacillus fraction of immunosuppression induced by cancer chemotherapeutic agents

Summary Contact hypersensitivity (CH) to 2,4-dinitro-1-fluorobenzene (DNFB) was induced in guinea pigs and mice by DNFB skin application. Development of CH was suppressed in both species either by cyclophosphamide (CY) treatment after sensitization or by single intravenous injection of dinitrobenzene-sulfonate (DNBS) before sensitization (hapten-induced tolerance). Additional treatment schedules were employed in guinea pigs, with the following results: Suppression of CH by injection of DNBS concomitant with sensitization; abrogation of hapten-induced tolerance by administration of CY before sensitization; and potentiation of CH skin reactivity by administration of CY before sensitization.Pretreatment by two injections of the methanol extraction residue (MER) tubercle bacillus fraction restored significantly the ability of CY treated animals to respond to DNFB sensitization. In contrast, administration of MER either by one injection before sensitization, concomitant with DNFB, or after sensitization did not prevent immunosuppression by CY.MER treatment was not effective in reversing hapten-induced tolerance in mice, and had only an occasional effect on this process in guinea pigs. Abrogation of hapten-induced tolerance and potentiation of DNFB sensitization by CY in guinea pigs were also not influenced by MER treatment.Supported by Contract NO1-CM-12127 from the NCI and by research grants from Concern Foundation, Inc., the Lautenberg Endowment, the National Council for Research and Development, Israel, and the GSF Munich, Germany, and the Leukemia Research Foundation, Inc.     

Prevention by the MER tubercle bacillus fraction of immunosuppression induced by cancer chemotherapeutic agents

Summary Contact hypersensitivity (CH) to 2,4 dinitro-1-fluorobenzene (DNFB) was induced in BALB/c mice by DNFB skin application. Development of skin CH was suppressed by exposure of the animals after sensitization to the cancer chemotherapeutic drugs cyclophosphamide (CY), sodium methotrexate (MTX), and 5-fluorouracil (5FU). Unresponsiveness to DNFB was also induced in parallel experiments by a single intravenous injection of dinitrobenzenesulfonate (DNBS), either before or concomitant with sensitization. Potentiation of CH skin reactivity was achieved by administration of CY prior to sensitization.Pretreatment by two injections of the methanol extraction residue (MER) tubercle bacillus fraction restored significantly the ability of animals exposed to CY, MTX, or 5FU to respond to DNFB sensitization. The agent did not impair the potentiation of CH skin reactivity that could be effected by administration of CY prior to sensitization.MER treatment was not effective in reversing hapten-induced (DNBS) tolerance in mice.These findings favor the assumption that MER, under the conditions tested, stimulates the function of positively reacting T cells and exerts no enhancing or protective action on suppressor T cells.     

Prevention by the MER tubercle bacillus fraction of immunosuppression induced by cancer chemotherapeutic agents

Summary Primary and secondary anti-sheep red blood cell antibody formation by BALB/c mice was reduced by administration of cyclophosphamide shortly following primary and secondary immunization. Treatment with the MER tubercle bacillus fraction by several schedules potentiated markedly and significantly the recovery of ability to respond to the antigen, as indicated by the production of specific plaque-forming cells and free hemagglutinating antibody.     

iASPPsv antagonizes apoptosis induced by chemotherapeutic agents in MCF-7 cells and mouse thymocytes

iASPP was an inhibitory member of ASPP family and could specifically inhibit the apoptotic function of p53. iASPPsv was identified by our lab as the short isoform of iASPP, which encoded a 407aa protein and highly matched the carboxyl terminus of iASPP. In this study, iASPPsv was stably transfected into the breast cancer cell line MCF-7 by means of lentivirus to explore the effects of iASPPsv on biological functions of MCF-7. Thymocytes from iASPP/iASPPsv transgenic mice were also used to explore the effects of iASPP/iASPPsv on cell biological function. The results demonstrated that iASPPsv antagonized the growth inhibition induced by etoposide (VP-16) in MCF-7 cells. iASPPsv also down-regulated proapoptotic genes (Bax, Puma and Noxa) expression to inhibit apoptosis caused by VP-16. Moreover, iASPP and iASPPsv could both help the thymocytes of transgenic mice to resist the growth inhibition and apoptosis caused by dexamethasone (Dex) or VP-16. At the same time, DNA double strand break damage accumulated in either iASPPsv MCF-7 cells or iASPP/iASPPsv thymocytes. These findings showed that iAPSS/iASPPsv reduced the growth inhibition and apoptosis induced by Dex or VP-16, with DNA damage accumulating which might promote the pathogenesis and/or progression of cancer.     

Effect of Human WEE1 and Stem Cell Factor on Human CD34^＋ Umbilical Cord Blood Cell Damage Induced by Chemotherapeutic Agents

Myelosuppression is one of the major side-effects of most anticancer drugs. To achieve myeloprotection, one bicistronic vector encoding anti-apoptotic protein human WEE l （WEElHu） and proliferation-stimulating stem cell factor （SCF） was generated. In this study, we selected human umbilical cord blood CD34^＋ cells as the in vitro model in an attempt to investigate whether WEEIHu, rather than conventional drug-resistant genes, can be introduced to rescue cells from the damage by chemotherapeutic agents such as cisplatin, adriamycin, mitomycin-c and 5-fluorouracil. Cell viability and cytotoxicity assay, colony-forming units in culture assay and externalization of phospholipid phosphatidylserine analysis showed that the expression of WEElHu and SCF in CD34^＋ cells provided the cells with some protection. These findings suggest that the expression of WEElHu and SCF might rescue CD34^＋ cells from chemotherapyinduced myelosuppression.     

Unbalanced growth and cell death in HeLa S3 cultures treated with DNA synthesis inhibitors

Flow cytometry indicated that significant amounts of dsRNA were accumulated in HeLa S3 cells blocked at or near G₁/S boundary by hydroxyurea (HU) or excess thymidine (TdR). The dsRNA/DNA ratio increased in these cells in a manner characteristic of unbalanced cell growth. In HU-treated cells, dsRNA content was maximal 16 hours after addition of the drug and did not change significantly during the next 24 hours. The DNA content in blocked cells increased by 10%. Cell viability assessed by colony formation in soft agar decreased exponentially in HU-treated cultures after 16 hours of incubation. Correlation between loss of cell viability and rate of cell proliferation after removal of HU was observed, as determined by cell count and analysis of cell cycle progression. In TdR-treated cultures cells slowly progressed into mid S-phase during 40 hours and dsRNA accumulation continued during this period. Cell viability was not significantly affected by treatment with excess TdR, indicating that unbalanced growth per se, as measured by dsRNA accumulation, is not lethal for the cells. After reversal of DNA synthesis inhibition by removal of the drug, cells treated with HU for 16 hours or TdR for 16–24 hours promptly progressed through the cell cycle. This progression was accompanied by accumulation of significant amounts of dsRNA. As a result, cells in G₂ phase had a very high dsRNA content leading to retention of the unbalanced condition (increased dsRNA/DNA ratio) in the daughter cells. It is suggested that dsRNA accumulation in the cell is controlled to a certain degree by cell progression through the S phase. This type of control, evidently, was reflected in limited dsRNA accumulation in the cells blocked at or near G₁/S border, in continuous dsRNA accumulation in the cells slowly progressing through S phase, and in accumulation of large amounts of dsRNA after renewal of progression through the S phase.     

Hepatocyte growth factor decreases sensitivity to chemotherapeutic agents and stimulates cell adhesion, invasion, and migration

Hepatocyte growth factor (HGF), also known as scatter factor (SF), plays an important role in cell:cell adhesion, cell proliferation, motility, and invasiveness of epithelial cells and tumor cells. In this study, we examined the effects of HGF on these types of biological activities and chemosensitivity in Chinese hamster ovary (CHO) cells by stable transfection of the HGF gene. HGF-transfected clones produced very high titers of HGF protein, whereas control vector-transfected clones did not produce detectable HGF protein. HGF-transfected clones showed modestly increased proliferation rates and became more resistant to cell death and apoptosis caused by two anticancer drugs, adriamycin (ADR) and camptothecin (CPT), compared to controlvector-transfected clones. Furthermore, HGF-transfected clones also exhibited increased activities of cell adhesion, migration, and invasion. The current study is the first demonstration that overexpression of the HGF gene affects chemosensitivity and cell metastasis behaviors, suggesting that HGF signaling pathway is a promising new target of therapeutic intervention of tumors.     

Benzyladenine-controlled protein synthesis and growth in apple cell suspensions

Prolific growth in cytokinin-requiring apple cell suspensions was achieved during 10 days of culture with appropriate concentrations of benzyladenine (BA). Unlike the controls, BA-treated cells showed a well developed sub-cellular organization and protein synthesis. Upon transfer to fresh cytokinin-free medium, however, these cells exhibited a rapid decrease in polyribosome formation, but with unchanged nuclear and nucleolar activities. Cells lacking added cytokinin showed a reduced metabolic activity and failed to divide. Deficiency in the translation process of cytokinin-deprived cells was overcome by exogenous BA-treatment. In cells maintained on a cytokinin-free medium for 5 days, BA-treatment enhanced protein synthesis from pre-existing mRNA in a specific way.     

KAP1 dictates p53 response induced by chemotherapeutic agents via Mdm2 interaction

KAP1 recruits many proteins involved in gene silencing and functions as an integral part of co-repressor complex. KAP1 was identified as Mdm2-binding protein and shown to form a complex with Mdm2 and p53 in vivo. We examined the role of KAP1 in p53 activation after the treatment of cells with different types of external stresses. KAP1 reduction markedly enhanced the induction of p21, a product of the p53 target gene, after treatment with actinomycin D or gamma-irradiation, but not with camptothecin. Treatment with actinomycin D, but not with camptothecin, augmented the interaction of p53 with Mdm2 and KAP1. Further, KAP1 reduction in actinomycin D-treated cells facilitated cell cycle arrest and negatively affected clonal cell growth. Thus, the reduction of KAP1 levels promotes p53-dependent p21 induction and inhibits cell proliferation in actinomycin D-treated cells. KAP1 may serve as a therapeutic target against cancer in combination with actinomycin D.     

Bone marrow osteoblast damage by chemotherapeutic agents

Hematopoietic reconstitution, following bone marrow or stem cell transplantation, requires a microenvironment niche capable of supporting both immature progenitors and stem cells with the capacity to differentiate and expand. Osteoblasts comprise one important component of this niche. We determined that treatment of human primary osteoblasts (HOB) with melphalan or VP-16 resulted in increased phospho-Smad2, consistent with increased TGF-β1 activity. This increase was coincident with reduced HOB capacity to support immature B lineage cell chemotaxis and adherence. The supportive deficit was not limited to committed progenitor cells, as human embryonic stem cells (hESC) or human CD34+ bone marrow cells co-cultured with HOB pre-exposed to melphalan, VP-16 or rTGF-β1 had profiles distinct from the same populations co-cultured with untreated HOB. Functional support deficits were downstream of changes in HOB gene expression profiles following chemotherapy exposure. Melphalan and VP-16 induced damage of HOB suggests vulnerability of this critical niche to therapeutic agents frequently utilized in pre-transplant regimens and suggests that dose escalated chemotherapy may contribute to post-transplantation hematopoietic deficits by damaging structural components of this supportive niche.