The role of transferrin receptors in hemopoietic cell growth

The Tf receptor is a prototypical cell surface nutrient receptor critical for growth of both normal and malignant blood cells. Because of the apparently universal requirement of growing blood cells for Fe, control of Tf receptor expression and function will continue to be a topic of considerable interest in experimental hematology and tumor cell biology. Studies of Tf receptor gene expression have already revealed unique patterns of gene regulation, and understanding control of Tf receptor gene expression during hemopoietic cell growth and differentiation may provide insights into factors controlling expression of other proliferation-related genes. Although much further study is required, early studies suggest that measuring serum Tf receptor and Tf-Tf receptor complexes may be a major advance in clinical assessment of erythropoiesis. Whether altering Tf receptor protein function or gene expression can be exploited to treat hematologic tumors remains unclear, but further studies are in progress.     

Characterization of acylfulvene histiospecific toxicity in human tumor cell lines

Purpose: Acylfulvene derivatives demonstrate marked efficacy in xenograft carcinoma models as compared with the parent illudin compounds. To elucidate the increased therapeutic efficacy of acylfulvene analogs, we compared them with the illudin compounds in terms of their in vitro cytotoxicity, cellular accumulation and DNA incorporation. Methods: The cytotoxicity of various acylfulvene analogs was tested in vitro against a variety of tumor cell lines. Radiolabelled acylfulvene analog was prepared and used for cellular accumulation and DNA incorporation studies. Results: The prototype acylfulvene analog retained selective histiospecific toxicity towards myeloid leukemia and various carcinoma cell lines. In vitro killing of tumor cells by acylfulvene required up to a 30-fold increase in molecules per cell, as compared with illudin S, indicating that acylfulvene was less toxic on a cellular level. At equitoxic concentrations, acylfulvene incorporation into genomic tumor cell DNA was equivalent to illudin S suggesting that cellular metabolism has a role in acylfulvene cytotoxicity. Analysis of cellular accumulation of acylfulvene into tumor cells revealed a markedly higher V_max for tumor cells, and a lower V_d for diffusion accumulation into other cells. Conclusions: The combination of higher V_max and lower V_d may explain the increased in vivo efficacy of acylfulvene. Received: 26 March 1997 / Accepted: 14 July 1997     

Preclinical re-evaluation of benzaldehyde as a chemotherapeutic agent

Benzaldehyde (BA), an agent extracted from figs, is reported to have antitumor activity in vitro against a variety of experimental tumors and in vivo against refractory human neoplasms. We employed recently developed in vitro techniques to examine the effect of BA on growth of malignant human cells, and examined its effects in vivo against two human tumor xenografts established from primary specimens. BA showed dose-dependent inhibition of HL60 promyelocytic leukemia cells and normal human granulocyte/macrophage colonies. It showed no in vitro activity against either KG-1 myeloid leukemia cells or chronic lymphocytic leukemia cells grown in colony culture. BA failed to inhibit growth of either HL60 or KG-1 cells in liquid culture, and did not induce differentiation of HL60 cells. When tested against human tumor colony-forming cells from 30 patients with solid tumors, inhibition of colony growth greater than 70% was seen in six patients (20%). BA failed to inhibit the in vivo growth of either the T222 epidermoid carcinoma xenograft or the T380 ovarian carcinoma xenograft. We conclude that BA lacks significant activity against most human tumors tested in these experimental systems.     

Chromosome abnormalities in malignant melanoma: clinical significance of nonrandom chromosome abnormalities in 206 cases

We report the cytogenetic abnormalities from a series of 206 primary malignant melanoma specimens referred to a single institution. A total of 169 out of 206 unique cases had chromosome breakpoints. A previously described statistical method was used to detect nonrandom distribution of chromosome breakpoints at the level of chromosome regions. Nonrandom occurrence of chromosome breakpoints (indicating that the observed number of breaks significantly exceeded the expected number of breaks) was detected in 28 regions, suggesting a hierarchy of genetic abnormalities in melanoma. Clinical variables and tumor characteristics were analyzed for associations with the presence of any nonrandom chromosome breakpoints; with individual, nonrandomly involved chromosome regions; and with paired, nonrandomly involved chromosome regions. No nonrandomly involved chromosome regions or pairs of regions appeared to significantly affect survival. These results identify recurring, nonrandom chromosome abnormalities in malignant melanoma. These results suggest that recurring, nonrandom chromosome alterations play a key role in the etiology and/or progression of malignant melanoma and identify targets within the genome for molecular genetic studies.     

Chromosome abnormalities in ovarian adenocarcinoma: II. Prognostic impact of nonrandom chromosome abnormalities in 244 cases

In a large series of ovarian carcinomas from 244 patients, 134 cases had chromosome rearrangements. We showed before that the pattern of chromosome breakpoints involved 21 separate chromosome regions nonrandomly and, in 90% of cases with breaks, the breakpoints occurred within 13 commonly involved regions. Log-rank and proportional hazards regression analyses showed that the aggregate presence of a chromosome breakpoint in any of 21 nonrandomly involved regions and breaks in 9 distinct regions (1p1, 1q2, 1p3, 3p1, 6p2, 11p1, 11q1, 12q2, and 13p1) were associated with reduced patient survival. Breakpoints in other areas of the genome, including other nonrandomly involved regions, were not associated with decreased survival. Because many cases had breakpoints in more than one nonrandomly involved region, proportional hazards regression was also used to analyze for effects of each nonrandomly involved region, controlling for effects of other regions. With this approach, only breakpoints within 1p1 and 3p1 retained independent, deleterious effects on survival. Similarly, when nonrandomly involved regions were entered into a proportional hazards model containing clinical variables associated with altered patient survival (tumor grade, tumor stage, and residual disease > 1 cm after resection), only 1p1 (P = 0.007) and 3p1 (P = 0.04) were associated with independent, negative effects on survival. These studies demonstrate that chromosome breakpoints within specific, nonrandomly involved chromosome regions are associated with impaired survival in ovarian cancers. Regions 1p1 and 3p1 are identified as areas of particular significance and are appropriate targets for analytical techniques such as SAGE and microarray analysis.     

Chromosome abnormalities in ovarian adenocarcinoma: I. Nonrandom chromosome abnormalities from 244 cases.

Cytogenetics provides important insights into the molecular pathogenesis of human cancers. Although extensive data exist on recurring cytogenetic abnormalities in hematologic cancers, data on individual solid tumor types remain limited. Previous studies of ovarian carcinoma indicated the presence of multiple, complex clonal chromosome abnormalities. Cytogenetics remains one of a few techniques capable of detecting these multiple, simultaneously occurring genetic abnormalities. We describe cytogenetic abnormalities from a series of 244 primary ovarian cancer specimens referred to a single institution. A total of 201/244 cases had fully characterized clonal chromosome abnormalities, of which 134 showed clonal chromosome breakpoints. We used a novel statistical technique to detect nonrandom chromosome breakpoints at the level of chromosome regions. Nonrandom occurrence of chromosome breakpoints was detected at regions 1p1*, 1q1*, 1p2*, 1q2*, 1p3*, 1q3, 3p1*, 1q4*, 6q1*, 6p2, 6q2, 7p1*, 7q1, 7p2*, 11p1*, 11q1, 11q2*, 12p1, 12q2*, 13p1, and 19q1. Simultaneous occurrence of multiple abnormalities was common. However, 120/134 cases had breakpoints at one or more of 13 commonly involved regions (*), suggesting a hierarchy of genetic abnormalities. Among clinical and tumor variables that predict patient survival, tumor grade was significantly associated with the presence of chromosome breakpoints. In additional studies, we show that nonrandom chromosome abnormalities are associated with impaired survival in ovarian cancer and that specific, nonrandomly involved chromosome regions retain significant effects on survival when analyses are controlled for important clinical variables. Additional specific chromosome abnormalities in this series are described, including chromosome gains and losses in near-diploid cases and homogeneously staining regions. These results suggest that recurring, nonrandom chromosome abnormalities are important in the pathogenesis and/or progression of ovarian cancers, and target areas of the genome for molecular genetic studies.     

Simple numeric abnormalities as primary karyotype changes in ovarian carcinoma

Simple near-diploid karyotypes in ovarian cancer may indicate either primary alterations related to tumor pathogenesis or abnormalities associated with early tumor progression. We have identified a series of 13 epithelial ovarian tumors with very simple karyotypes. Specifically, these karyotypes were near-diploid and displayed numeric abnormalities alone or combined with one or two structural alterations. The present series includes samples from 10 patients with newly diagnosed adenocarcinomas and 3 patients having borderline malignancies. Recurrent numeric abnormalities were identified and included 9/13 cases (69%) with + 12, eight cases (62%) with + 8, five cases (38%) with + 7, three cases (23%) each with + 3 or + 5, and two cases (15%) with -X, Five cases in this series displayed certain numeric abnormalities (+ 12, + 7, and -X) as the sole anomalies, thereby qualifying as primary karyotype changes. Of the 6 cases with structural abnormalities, 4 involved chromosome 19, 2 involved chromosome I, and the remaining abnormalities or translocation partners involved other chromosomes. These findings indicate that some numeric abnormalities are primary karyotype alterations in patients with malignant epithelial ovarian tumors and that chromosome 19 may be preferentially involved in structural rearrangements during early tumor progression. Genes Chromosom Cancer 10:262–266 (1994). © 1994 Wiley-Liss, Inc.     

Modulation of leukaemia blast colony growth by steroid hormones

The effect of steroid hormones on in vitro colony growth of human myelogenous leukaemia cells was examined. β-Oestradiol, testosterone and 5-β-dihydrotesterone had little effect on HL60 promyelocytic leukaemia cells or blast colony forming cells (CFC) from eight patients with acute non-lymphocytic leukaemia (ANLL). These compounds inhibited blast colony growth at very high concentrations (10⁻⁶-10⁻⁴ M). In contrast, hydrocortisone (HC) had highly variable effects on blast colony growth. HL60 cells were resistant to HC at concentrations up to 10⁻⁴ M but blast CFC showed a wide range of response. Some patients were resistant to HC at all concentrations tested, while others were inhibited by concentrations as low as 10⁻⁸ M. The inhibitory effect of HC was also observed on ³H-TdR incorporation by stimulated peripheral blood blasts. Inhibition by HC was not blocked by progesterone, suggesting this effect was not mediated by specific hormone receptors. These data indicate differences in the response of blast cells in ANLL and normal progenitors to steroid hormones. Cells from patients with ANLL display variable inhibition by HC which is probably not mediated by specific receptors.     

Effects of insulin and insulin-like growth factor I on growth of human leukemia cells in serum-free and protein-free medium

Human myeloid leukemia cells (HL60) and malignant lymphocytes (Namalwa) were grown in protein-free, Fe-supplemented media and used to study growth responses to insulin and insulin-like growth factor 1 (IGF-I). HL60 cells previously grown in serum-free medium containing microgram quantities of insulin showed an 18-fold reduction in cumulative cell production when grown without insulin. However, the same cells showed reduced or absent growth stimulation with 1 to 100 ng/mL insulin or IGF- I for at least four days following insulin deprivation, indicating that culture conditions modified insulin and IGF-I responses. When the same cells were grown in Fe-supplemented, protein-free medium (RPMI-Fe), insulin and IGF-I caused dose-dependent stimulation of HL60 cell growth with half-maximal stimulation at nanogram concentrations. Namalwa cells grown in protein-free medium showed no response to either hormone. Radioligand binding showed the presence of insulin and IGF-I receptors on both HL60 and Namalwa cells grown in RPMI-Fe. HL60 cells grown in fetal bovine serum had higher, and cells grown with microgram quantities of insulin dramatically reduced, insulin binding. Competitive binding studies and cultures with anti-IGF-I receptor antibody showed insulin and IGF-I stimulated growth through their respective specific receptors. Both insulin and IGF-I stimulate growth of some cultured human leukemia cells, but the presence of insulin or IGF-I receptors alone does not predict growth responses. Culture conditions affect both cellular responses and ligand binding by these hormones and must be closely controlled to study growth responses.