Benjamin Y. Klein

Structurally different bisphosphonates exert opposing effects on alkaline phosphatase and mineralization in marrow osteoprogenitors.

Bisphosphonates (BPs) are inhibitors of bone resorption and soft tissue calcification. The biological effects of the BPs in calcium‐related disorders are attributed mainly to their incorporation in bone, enabling direct interaction with osteoclasts and/or osteoblasts through a variety of biochemical pathways. Structural differences account for the considerable differences in the pharmacological activity of BPs. We compared the effects of two structurally different compounds, alendronate and 2‐(3′‐dimethylaminopyrazinio)ethylidene‐1,1‐bisphosphonic acid betaine (VS‐6), in an osteoprogenitor differentiation system. The BPs were examined in a bone marrow stromal‐cell culture system, which normally results in osteoprogenitor differentiation. The drugs were present in the cultures from days 2 to 11 of osteogenic stimulation, a period estimated as being comparable to the end of proliferation and the matrix‐maturation stages. We found that the two different BPs have opposing effects on specific alkaline phosphatase (ALP) activity, on stromal‐cell proliferation, and on cell‐mediated mineralization. These BPs differentially interact with cell‐associated phosphohydrolysis, particularly at a concentration of 10−2 of ALP Km, in which alendronate inhibits whereas VS‐6 did not inhibit phosphatase activity. VS‐6 treatment resulted in similar and significantly increased mineralization at 10 and 1 μM drug concentrations, respectively. In contrast, mineralization was similar to control, and significantly decreased at 10 and 1 μM drug concentrations, respectively, under alendronate treatment. J. Cell. Biochem. 68:186–194, 1998.

Changes related to phosphatidylinositol 3-kinase/Akt signaling in leiomyomas: possible involvement of glycogen synthase kinase 3α and cyclin D2 in the pathophysiology

OBJECTIVE To identify changes in the expression and phosphorylation of phosphatidylinositol 3-kinase (PI3K)/Akt protein kinases controlling survival and/or apoptosis of in vitro cell cultures of uterine leiomyomas. DESIGN Establishment of paired cell cultures of leiomyoma and myometrial specimens. SETTING Hadassah gynecology research laboratory. PATIENT(S) Eleven white premenopausal women, 35 to 50 years of age, undergoing hysterectomy because of symptomatic uterine leiomyomas. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Immunochemical analysis of expression and phosphorylation of relevant PI3K/Akt and BCL2 proteins. RESULT(S) Analysis of total phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and of nonphosphorylated and phosphorylated (p) PDK1, Akt, glycogen synthase kinase 3 (GSK3), FKHR, tuberin (TSC2) and hamartin (TSC1) complex, and cyclin D2 proteins indicated that [1] the level of pGSK3alpha and cyclin D2 proteins was elevated significantly in the leiomyoma compared with the normal myometrium, [2] there was a significant interaction between PTEN- PDK1 and between pAkt-pGSK3beta in the leiomyoma compared with the myometrial cells, and [3] there was a significant interaction between pAkt-pGSK3alpha in the paired leiomyoma and myometrial cultures. CONCLUSION(S) Our study suggests that the downstream signaling components of the PI3K/Akt pathway, GSK3 (a regulator of apoptosis), and cyclin D2 (a promoter of G1/S progression), as well as the significant interaction between PTEN-PDK and between pAkt-pGSK3beta, are involved in the survival and proliferation of leiomyomas.

Induction of Antitumor Reactive Cells or Suppressor Cells by Different Molecular Species Isolated from the Same Nonimmunogenic Tumor

The inability of spontaneous and some laboratory-induced tumors to stimulate the immune system has continuously raised the question of the validity of using immunological maneuvers in order to control tumor growth. In this project we suggest that a tumor which is nonimmunogenic still has an immunogenic potential that can be revealed and used in order to stimulate antitumor immunity and consequently tumor destruction. YAC, a Moloney-virus-induced tumor of A mice, failed to stimulate immunological responses. This tumor homogenate was exposed to nonreduced sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). At the end of the electrophoresis, the gels were sliced and injected in sequential order into various groups of A mice. It was found that some of the gel slices (usually with M.W. of 100 K or less) induced cytotoxic responses, whereas other gel slices (usually with M.W. of about 150 K) induced suppressor cells. Similarly, certain gel slices induced cells that inhibited the in vivo tumor growth, whereas others enhanced the in vivo tumor growth. These last two types of cells did not present the same cellular population that mediated the cellular cytotoxicity or the suppressive effects respectively. It was concluded that poorly immunogenic tumor cells do possess immunogenic potential that can be revealed after dissociation between the immunogenic and suppressogenic entities.

Acetaldehyde differentially affects the growth of uterine leiomyomata and myometrial cells in tissue cultures

OBJECTIVE To examine the effect of alcohol and its derivatives on leiomyomata versus normal myometrium in cell culture. DESIGN A study on human tissue cultures. SETTING A tertiary-care university hospital. PATIENT(S) Premenopausal women with uterine myomas. INTERVENTION(S) Obtaining paired cultures of normal myometrium and myomas from women undergoing hysterectomy. MAIN OUTCOME MEASURE(S) Analysis of the effect of ethanol and acetaldehyde on cell growth and the expression of relevant proteins. RESULT(S) Acetaldehyde statistically significantly inhibited the growth of myoma cells compared with normal myometrium. The level of alcohol dehydrogenase 1 (ADH1) protein was lower in myoma than in myometrial cells. The acetaldehyde dehydrogenase 1 (ALDH1) protein level was higher in myoma cells. Treatment with acetaldehyde resulted in a relative reduction of ALDH1 level in the myoma cells. CONCLUSION(S) Acetaldehyde has an inhibitory effect on cell growth of myoma compared with normal myometrium. The reduced level of ADH1 and the increased level of ALDH1 proteins observed in myoma cell culture reduces the acetaldehyde level and thus may be involved in myoma cell growth.

Immunogenicity of subcellular fractions and molecular species of MuLV-induced tumors. I. screening of immunogenic components by isopycnic ultracentrifugation and polyacrylamide electrophoresis of a tumor homogenate

Abstract The isolation of immunogenic determinants from subcellular fractions of tumor cells by a biochemical screening method is described in this paper. Separated subcellular fractions and molecular species of tumor cells were screened for their ability to stimulate antitumor immune responses in syngeneic hosts. Homogenates of the in vivo-carried tumor YAC of A/J mice and the corresponding in vitro-cultivated tumor YAC-1 were fractionated on a sucrose gradient. The immunogenic capacity of the fractions was screened by injecting each fraction into A/J mice and testing the ability of the splenocytes of these mice to generate anti-YAC cytotoxic responses. The strongest cytotoxic responses were generated by the heavy subcellular fractions of YAC and YAC-1, which contained the highest concentration of protein, and the light fraction of YAC-1, which contained high 5′-nucleotidase activity. These immunogenic fractions and homogenates of intact tumor cells were further analyzed by sodium dodecyl sulfate-polyacrylamde gel electrophoresis (SDS-PAGE). The immunogenicity of the molecular species separated by SDS-PAGE was tested by injecting the gel slices into different groups of mice. It was found that certain molecular species stimulated specific antitumor cytotoxic responses, whereas others failed to stimulate such responses. Mice injected with some of these immunogenic molecular species were able to reject viable tumors. Other immunogenic molecular species, although stimulating cellular cytotoxic responses, failed to generate rejection capacity in the syngeneic hosts. To the best of our knowledge this is the first attempt to isolate immunogenic determinants from tumor cells by a biochemical screening method. Furthermore, by this method evidence was obtained that immunogenic determinants can even be isolated from a non-immunogenic tumor.

All-trans-retinoic acid mediates changes in PI3K and retinoic acid signaling proteins of leiomyomas

OBJECTIVE To detect changes induced by all-trans-retinoic acid (ATRA) on the expression and activation of target proteins of the retinoic acid (RA) and PI3K/Akt pathways involved in leiomyoma growth. DESIGN A study on human tissue cultures. SETTING Hadassah University Hospital. PATIENT(S) Premenopausal women with uterine leiomyomas. INTERVENTION(S) Paired cultures of normal myometrium and leiomyomas, from women undergoing hysterectomy, were obtained. MAIN OUTCOME MEASURE(S) The effect of ATRA was examined on the expression and phosphorylation of relevant RA, PI3K/Akt, and Bcl2 proteins (immunochemical analysis), cell proliferation, cell cycle distribution, and apoptosis. RESULT(S) Applying our cell culture model, we demonstrated that ATRA induced changes in the expression and activation of the RA and PI3K/Akt pathway proteins in leiomyoma cells, with significant increases of alcohol dehydrogenase 1 and cyclin D2 protein levels. In part of the leiomyoma cells, ATRA induced a relative increase of Bax (proapoptotic) as well as a relative decrease of phosphorylated glycogen synthase kinase 3β (proapoptotic). CONCLUSION(S) Our results highlight the involvement of ATRA in the RA and PI3K/Akt pathways, whose specific signaling products may influence the outcome of leiomyoma growth by regulating cell proliferation, apoptosis, and survival. These results might be useful for the on-going research into alternative methods for treating and preventing this disorder.

LDL induces Saos2 osteoblasts death via Akt pathways responsive to a neutral sphingomyelinase inhibitor

Atherosclerosis is epidemiologically associated with postmenopausal osteoporosis (OP) presumably by common etiologic factors, reflecting a state of co‐morbidity in aging. Osteoblasts make a significant facet of this co‐morbidity state. Since oxidized low‐density lipoprotein (oxLDL) is a major factor in generation of vascular wall pathology, we examined the ability of native LDL (nLDL) and oxLDL to induce Saos2 osteoblasts growth arrest. OxLDL induced Saos2 cell death with morphological features of apoptosis that was inhibited mainly by caspase‐9 and partially by caspase‐3 but not by caspase‐8 inhibitors. nLDL, like oxLDL, has induced cell death, where 60% (P = 0.00033) and 30% (P = 0.075, ns) of the cell death, respectively, could be inhibited by scyphostatin (a neutral sphingomyelinase [nSMase] inhibitor). Upon similar condition, nLDL inhibited the phosphorylation of Akt and two of its downstream targets, fork head receptor (FKHR) and glycogen synthase kinase‐3 (GSK3). This is a pathway that stimulates cell survival and proliferation. nLDL has also induced an increase in the proapoptotic Bcl‐Xs and it has diminished the potential antiapoptotic Src kinase activity. At the 4 h time‐point, upon a substantial decrease in nLDL‐induced Akt phosphorylation, scyphostatin has inhibited the reduction in FKHR and GSK3 phosphorylation but inexplicably not that of Akt. Scyphostatin has also corrected the reduction in Src kinase activity. Taken together, the results indicate that nLDL has induced apoptosis in Saos2 osteoblasts by inactivation of the pathway downstream to Akt using nSMase, and by involvement of Src kinase. Inferring that caspase‐9 was the main executioner (rather than caspase‐8 and‐3) in Saos2 cell death, indicates that the nSMase‐induced release of ceramide, directly activated the intrinsic mitochondrial apoptotic pathway. With regard to the Akt inactivation by nLDL, Saos2 osteoblasts responded in an opposite fashion to the response reported by others, in macrophages. J. Cell. Biochem. 98: 661–671, 2006.

Family history of uterine fibroids associated with low level of fumarate hydratase in leiomyomata cells.

Despite the tremendous impact of uterine fibroids on womens’ quality of life and health economy, the mechanism of their development remains an enigma. Several mechanisms have been suggested, including endocrine, paracrine and genetic theories. A number of lines of evidence support a genetic basis for uterine fibroid formation. These include ethnic variation, familial predisposition, twin studies and unique familial syndromes. In 1973 Reed et al. described a syndrome of familial cutaneous and uterine leiomyomata [1]. Tomlinson et al. have demonstrated that germ-line mutations in the gene for fumarate hydratase (fumarase), a Krebs-cycle enzyme, predispose to dominantly inherited uterine fibroids, skin leiomyomata and papillary renal cell carcinoma [2]. Recently, it has been shown that fumarate hydratase mutations are involved also in nonsyndromatic uterine leiomyomas [3]. Given these genetic data, we set out to measure the level of this enzyme, at the protein level, in leiomyoma and in normal myometrial tissues and to correlate its levels with family history. We hypothesized that family history is inversely related to fumarate hydratase levels in the myoma cells. After obtaining IRB approval and informed consent as required, paired cell cultures of leiomyoma and adjacent normal myometrium tissue samples were obtained from hysterectomy specimens of 14 pre-menopausal women with a symptomatic fibroid uterus. Our techniques for myoma/ myometrium cell cultures have been described in detail elsewhere [4]. Fumarate hydratase levels in leiomyoma and myometrium were analyzed by Western blot and compared with family history of symptomatic fibroids. Clinical data were obtained from patient’s charts and telephone interviews. Significant family history of fibroids was defined as at least one first-degree relative with symptomatic uterine fibroids requiring surgery. Descriptive and univariate analyses were used to determine statistical significance (p < 0.05). Our results showed that patients who had positive family history of symptomatic fibroids had significantly lower levels of fumarate hydratase in the leiomyoma cells compared to the myometrium (p = 0.011, Fig. 1). There were no differences in patients’ demographics, clinical presentation and uterine size between the groups. The suggested mechanism for tumorigenesis attributed to the mutation in fumarate hydratase is pseudo-hypoxic drive. Fumarate hydratase is a Krebs-cycle enzyme that converts fumarate to malate. Fumarate hydratase-deficient cells accumulate fumarate which results in up-regulation of hypoxia-induced factor 1 alpha (HIF1a). The raised level of HIF1a results in increased expression of HIF-target genes, such as vasculo-endothelial growth factor (VEGF) and other genes related to angiogenesis. In normal conditions HIF1a is labile due to proteosomal degradation by HIF propyl hydroxylase. Under hypoxemia, this enzyme is inactivated and HIF1a is stabilized and activated. Thus, fumarate hydratase acts as a tumor suppressor and its inhibition may be associated with the development of tumors [5]. Despite the limitations of this study – its small sample size and retrospective design – these preliminary results suggest that positive family history of uterine fibroids is associated with low levels of fumarate hydratase in the myoma cells. Future prospective and adequately powered studies are needed to further support this hypothesis.

Immunogenicity of subcellular fractions and molecular species of MuLV-induced tumors. III. Stimulation of syngeneic antitumor responses by subcellular fractions and molecular species of Moloney virus-induced tumors in CBA and A mice.

YBA, a Moloney virus-induced leukemia in CBA mice, and a relatively weak immunogenic tumor, was screened for the presence of immunogenic antigens. The tumor was subjected to homogenization and subcellular fractionation on sucrose gradients; the immunogenic subcellular fractions underwent further separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The immunogenicity of the subcellular fractions and the SDS-PAGE-isolated molecular species were tested by (their) subcutaneous injection into syngeneic mice and examination of their splenocytes examined against tumor cell and normal cell targets by the chromium release cell-mediated lympholysis assay. Tumor cell homogenates were also separated by SDS-PAGE and tested for immunogenicity without prior fractionation. Splenocytes from mice that had received injections of certain SDS-PAGE-isolated epitopes derived from YBA tumor homogenate or its light and heavy subcellular fractions generated effective cytotoxic responses against YBA target cells after 6 days in vitro cultivation. In contrast, intact YBA tumor cells or non-separated tumor homogenates failed to induce an efficient cytotoxic response. The effector cells induced with the immunogenic SDS-PAGE-isolated epitopes of YBA tumor were specific, since they cytolysed the homologous target cells more efficiently than unrelated target cells or syngeneic normal cells. The activity of these effector cells was affected by varying the effector: target ratio. Augmentation of the cytotoxic responses was obtained when the splenocytes of mice immunized with SDS-PAGE-isolated epitopes of YBA tumor were restimulated in vitro, with the homologous neoplastic cells. Immunogenic SDS-PAGE epitopes were isolated from YAC tumor also (YAC is a Moloney-induced tumor of A mice). The effector cells induced with these separated epitopes were characterized as thymus-derived cells and not as natural killer cells. The results suggest that (1) the molecular repertoire of YBA and YBA tumors contain immunogens that can induce a specific antitumor cell-mediated response; (2) the isolated molecular species injected are more efficient immunogens than the entire, unseparated homogenate sample or a dose of 10(8) intact inactivated tumor cells; and (3) the gel matrix may be responsible for the enhanced cell-mediated response induced against the weakly immunogenic tumor.

A suggested mechanism for changing tumor cell phenotype: transfection of host cells with DNA sequences of dead tumor cells.

In observing the phenomenon of alterations of tumor cell phenotypes, one may envision a mechanism that induces the change in tumor cell characteristics and the appearance of metastasis. Schirrmacher (1) has suggested that the effect of the microenvironment on tumor cells influences the control of gene expression and that together with a selective process it may result in a newly arising phenotype. I suggest that in certain tumors the phenotypic change is the result of transfection of a mitotically active cell with certain DNA sequences arising from the tumor cells. Indirect evidence of support this suggested mechanism may be found in experimental results either interpreted differently or that happened to be by-products of experiments designed for other purposes. The appearance of tumors that acquire the allogeneic markers of their new allogeneic hosts suggests in vivo transfection with transforming DNA sequences (2, 4, 5, 6 7, 8). Experimental transfection of cells with DNA sequences resulting in transformation (19) strengthens this hypothesis. According to this hypothesis the immune system has a dual role in tumor alteration, it carries out the selection and it also induces the transformation of future new tumor phenotypes due to its potential to increase the availability of DNA breaks in the microenvironment.