Tumor Cell Dedifferentiation Research Articles

Thioredoxin reductase as a novel molecular target for cancer therapy

Tumor cell proliferation, de-differentiation, and progression depend on a complex combination of altered cell cycle regulation, excessive growth factor pathway activation, and decreased apoptosis. The understanding of these complex mechanisms should lead to the identification of potential targets for therapeutic intervention. Redox-sensitive signaling factors also regulate multiple cellular processes including proliferation, cell cycle, and pro-survival signaling cascades, suggesting their potential as molecular targets for anticancer agents. These observations suggest that redox-sensitive signaling factors may be potential novel molecular markers. We hypothesized that thioredoxin reductase-1 (TR), a component of several redox-regulated pathways, may represent a potential molecular target candidate in response to agents that induce oxidative stress. There have been numerous biological studies over the last decade investigating the cell biological, biochemical, and genetic properties of TR both in culture and in in vivo models. In addition, using a series of permanent cell lines that express either a wild-type TR or a dominant mutant TR gene or a chemical agent that inhibits TR we demonstrated that TR meets most criteria that would identify a molecular target. Based on these results we believe TR is a potential molecular target and discuss potential clinical possibilities.

Dedifferentiated liposarcoma with extensive lymphoid component

An unusual variant of dedifferentiated liposarcoma with extensive lymphocytic component is described. A 71-year-old patient suffered from a relapse of an atypical lipomatous tumor/well-differentiated liposarcoma with early micronodular (low-grade) dedifferentiation, which had been resected 4 years before. The relapse revealed features of a dedifferentiated liposarcoma with spindle-cell, partly pleomorphic dedifferentiation and osseous metaplasia. Clearly separated from the spindle-cell areas, an extensive homogeneously dense lymphoid (lymphocytic) tumor-component was evident, with relative abrupt transition to the well-differentiated liposarcoma component. Using immunohistochemistry and PCR, the lymphoid (“lymphoma-like”) infiltrate proved to be a polyclonal lymphocytic proliferation. Fluorescence in situ hybridization (FISH) analysis revealed no signs of MDM2- and CDK4-gene amplification in the lymphoid areas, although within this mononuclear lymphoid population, large polymorphic nuclei displayed an amplified number of MDM2/ CDK4 gene copies, indicating the presence of truly dedifferentiated tumor cells within the lymphoid component. The results favor a reactive lymphocytic infiltration versus a neoplastic one, which might be caused for example by chemoattractive agents. An extensive lymphoid “overgrowth” must be considered within the spectrum of unusual variants and in the differential diagnosis of dedifferentiated liposarcoma.

Hypoxia-induced dedifferentiation of tumor cells – A mechanism behind heterogeneity and aggressiveness of solid tumors

Histopathological examination of solid tumors frequently reveals pronounced tumor cell heterogeneity with regards to cell organization, cell morphology, cell size, nuclei morphology, etc. Analyses of gene expression patterns by immunohistochemistry or in situ hybridization techniques further strengthen the actual presence of phenotypic heterogeneity, often demonstrating substantial diversity within a given tumor. The molecular mechanisms underlying the phenotypic heterogeneity are very complex with genetic, epigenetic and environmental components. Hypoxia, shortage in oxygen, greatly influences cellular phenotypes by altering the expression of specific genes, and is an important contributor to intra- and inter-tumor cell diversity as revealed by the pronounced but non-uniform expression of hypoxia-driven genes in solid tumors (reviewed in [Semenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer 2003;3:721-32; Harris AL. Hypoxia--a key regulatory factor in tumour growth. Nat Rev Cancer 2002;2:38-47.]). The oxygen pressure in solid tumors is generally lower than in the surrounding non-malignant tissues, and tumors exhibiting extensive hypoxia have been shown to be more aggressive than corresponding tumors that are better oxygenized [Vaupel P. Oxygen transport in tumors: characteristics and clinical implications. Adv Exp Med Biol 1996;388:341-51; Vaupel P, Thews O, Hoeckel M. Treatment resistance of solid tumors: role of hypoxia and anemia. Med Oncol 2001;18:243-59.]. We recently observed that hypoxic neuroblastoma cells and breast cancer cells lose their differentiated gene expression patterns and develop stem cell-like phenotypes [Jögi A, Øra I, Nilsson H, Lindeheim A, Makino Y, Poellinger L, et al. Hypoxia alters gene expression in human neuroblastoma cells toward an immature and neural crest-like phenotype. Proc Natl Acad Sci USA 2002;99:7021-6; Helczynska K, Kronblad A, Jögi A, Nilsson E, Beckman S, Landberg G, et al. Hypoxia promotes a dedifferentiated phenotype in ductal breast carcinoma in situ. Cancer Res 2003;63:1441-4.]. As low stage of differentiation in neuroblastoma and in breast cancer is linked to poor prognosis, hypoxia-induced dedifferentiation will not only contribute to tumor heterogeneity but could also be one mechanism behind increased aggressiveness of hypoxic tumors. The effect(s) of hypoxia on tumor cell differentiation status is the focus of this review.

Role of Osteoclasts in Regulation of Human Hematopoietic Stem Cell Niche and Fate.

Recent studies indicate that osteoblasts play an important role in maintaining hematopoietic stem cells (HSCs) niche in the bone marrow microenvironment. The aim of study was to test the effect of osteoclasts on the fate of HSCs in a long term co-culture assay. To generate osteoclasts, peripheral blood mononuclear cells from mobilized donors were cultured for 6–10 days in αMEM media supplemented with 10% FCS, M-CSF and RANKL. After removal of non-adherent cells, the cultures contained 95% multinucleated osteoclasts and their precursors. These osteoclasts expressed TRAP and formed resorption pits on bone slices (Yaccoby et al., Cancer Res., 2004). CD34+ cells were purified from donor PBSCs and cord blood using immunomagnetic beads separation (>95% purity). Adult and cord blood HSCs were co-cultured with osteoclasts for up to 3 and 10 months, respectively, in media lacking any cytokines. Because osteoclasts do not survive long without M-CSF and RANKL, the HSCs were transferred to fresh osteoclast cultures every 6–10 days. Unlike their tight adherence to stromal cells, HSCs did not adhere to the osteoclasts and were easily recovered from co-cultures by gentle pipetting. Following 1 to 3 weeks of co-culture, committed HSCs rapidly differentiated into various hematopoietic cell lineage, followed by phagocytosis of terminal differentiated hematopoietic cells by the osteoclasts. The remaining HSCs were highly viable (>90% by trypan blue exclusion) and gradually lost their CD34 expression, so that the cultures contained subpopulations of HSCs expressing CD34−/lowCD38+ and CD34−/lowCD38−. Quantitive real time RT-PCR (qRT-PCR) revealed loss of expression of CD34 and reduced expression of CD45 by HSCs co-cultured with osteoclasts longer than 6 weeks. Variable expression of CD34 on HSCs was previously reported in murine but not human HSCs (Tajima et al., Blood, 2001). The co-cultured HSCs showed reduced capacity of generating in vitro hematopoietic colonies, and did not differentiate into osteoclasts upon stimulation with M-CSF and RANKL. We next tested the long term engraftment of these co-cultured HSCs in 2 animal models. In the first model, cord blood and adult HSCs from 2 donors recovered after >6 weeks in co-culture were injected I.V. into irradiated NOD/SCID mice. In the second novel model, co-cultured cord blood and adult HSCs from 2 donors were injected directly into rabbit bones implanted subcutaneously in SCID mice (SCID-rab model), 6–8 weeks after rabbit bone implantation. After 2–4 months, 10%±3% human CD45-expressing cells were identified in the NOD/SCID mice femora and 8%±4% in the SCID-rab mice rabbit bone. Our study suggests that osteoclasts promote rapid differentiation of committed HSCs and induce conversion of CD34+ cells to CD34− stem cells with self renewal potential. Intriguingly, long term co-culture of primary CD138-selected myeloma plasma cells (n=16) with osteoclasts resulted in dedifferentiation of tumor cells from a mature CD45− phenotype to an immature, CD45-expressing cells, suggesting a common mechanism of osteoclast-induced HSC and myeloma cell plasticity. This indicates that osteoclasts are important bone marrow component regulating human HSC niche, plasticity and fate.

Loss of ERbeta expression as a common step in estrogen-dependent tumor progression.

The characterization of estrogen receptor beta (ERbeta) brought new insight into the mechanisms underlying estrogen signaling. Estrogen induction of cell proliferation is a crucial step in carcinogenesis of gynecologic target tissues, and the mitogenic effects of estrogen in these tissues (such as breast, endometrium and ovary) are well documented both in vitro and in vivo. There is also an emerging body of evidence that colon and prostate cancer growth is influenced by estrogens. In all of these tissues, most studies have shown decreased ERbeta expression in cancer as compared with benign tumors or normal tissues, whereas ERalpha expression persists. The loss of ERbeta expression in cancer cells could reflect tumor cell dedifferentiation but may also represent a critical stage in estrogen-dependent tumor progression. Modulation of the expression of ERalpha target genes by ERbeta or ERbeta-specific gene induction could explain that ERbeta has a differential effect on proliferation as compared with ERalpha. ERbeta may exert a protective effect and thus constitute a new target for hormone therapy, such as ligand specific activation. The potential distinct roles of ERalpha and ERbeta expression in carcinogenesis, as suggested by experimental and clinical data, are discussed in this review.

Expression and localization of membrane-type-1 matrix metalloproteinase, CD 44, and laminin-5?2 chain during colorectal carcinoma tumor progression

Membrane-type-1 matrix metalloproteinase (MT1-MMP) is overexpressed in many malignant tumor tissues and would be involved in tumor-cell migration. Using dual immunofluorescence of frozen sections, this study examined the expression and localization of MT1-MMP and its interacting molecules, CD44 and laminin-5gamma2 chain (LN-5gamma2) monomer, in 48 cases of colorectal tumors. Recent studies have shown that MT1-MMP, CD44 and LN-5gamma2 are direct downstream targets in the adenomatosis polyposis coli (APC)/beta-catenin (Wnt)-signaling pathway, which is upregulated in most colorectal epithelial tumors. MT1-MMP overexpression was observed in adenocarcinoma cases with moderate and/or less differentiation coinciding with CD44 downmodulation. Recent observations indicate that MT1-MMP overexpression disrupts tubulogenesis of MDCK cells in type-I collagen-rich tissues. Therefore, MT1-MMP overexpression might involve disturbances of neoplastic glandular structures during colorectal adenocarcinoma tumor progression. Intensity distribution analyses of images with dual immunofluorescence indicated that overexpressed MT1-MMP is closely associated with the enhanced expression of the LN-5gamma2 monomers at the invasive front of dedifferentiated tumor cells. Additionally, the graded expression of nuclear active beta-catenin was found in moderately differentiated and dedifferentiated areas of adenocarcinomas, where MT1-MMP overexpression was observed. Therefore, this study reveals that MT1-MMP might be a major effector of Wnt signaling in the late stage of colorectal carcinoma tumor progression.

Shift of syndecan-1 expression from epithelial to stromal cells during progression of solid tumours

Syndecan-1 (SDC-1), a protein found on cells and in the extracellular matrix, participates in cell proliferation, cell migration and cell–matrix interactions. SDC-1 expression correlates with the maintenance of epithelial morphology and inhibition of invasiveness. In the present study, a second SDC-1 mRNA isoform was identified and the expression of both transcripts was investigated in various normal and malignant tissues. Both transcripts were coexpressed at equal levels in all tissues and organs analysed. Cancer-profiling array (CPA) analysis of 241 non-enriched tumour and normal cDNAs revealed stronger upregulation of SDC-1 in tumour tissues as compared with oligonucleotide array-based expression analysis of SDC-1 in microdissected breast, prostate, lung, and colon carcinoma cells. With in situ hybridisation and immunohistochemistry it was demonstrated that this difference in SDC-1 expression originates from stromal cells present in tumour connective tissue. But only the cells in connective tissue surrounding breast, lung, colon and bladder carcinoma showed upregulation of SDC-1. These stromal cells were characterised as spindle cells with myofibroblastic differentiation and they may contribute to the dedifferentiation of tumour cells and the development of metastasis.

Induction of aneuploidy by increasing chromosomal instability during dedifferentiation of hepatocellular carcinoma.

To gain more insight into the role of chromosomal instability (CIN), the cytogenetic hallmark of most solid tumors, we performed fluorescence in situ hybridization (FISH) on interphase nuclei of cytological specimens enabling the correct detection of chromosome copies in intact tumor cells of 18 well (G1), moderately (G2), or poorly (G3) differentiated hepatocellular carcinomas (HCCs). A close correlation between the morphological dedifferentiation and increasing copy numbers and variation of FISH signals was seen for chromosomes 1 and 8, respectively (P < or = 0.0002). Four HCC G1 had constant chromosome patterns for chromosomes 1 and/or 8 with a mean of signals per nucleus < or =5.08 and < or =3 different signal combinations, indicating a low level of CIN, as confirmed by FISH using probes for centromeres of chromosomes 3, 7, and 17. In contrast to this, five HCC G2-3 revealed > or =8.46 signals per nucleus and 23-41 different signal combinations, indicating high levels of CIN. In the remaining cases, signal counts from 5.96-8.46 and 7-15 combinations were seen. Here, nuclei with constant aberration patterns and low copy numbers occurred alongside nuclei with inconstant patterns and high copy numbers. It is evident that in these cases a transition from well to moderately differentiated HCC developed in parallel to an increase in CIN, possibly induced by a major dysregulation of mitotic control mechanisms. In conclusion, CIN may induce a stepwise increase of aneuploidy in HCC that is mirrored by the morphological dedifferentiation of tumor cells.

Induction of aneuploidy by increasing chromosomal instability during dedifferentiation of hepatocellular carcinoma

To gain more insight into the role of chromosomal instability (CIN), the cytogenetic hallmark of most solid tumors, we performed fluorescence in situ hybridization (FISH) on interphase nuclei of cytological specimens enabling the correct detection of chromosome copies in intact tumor cells of 18 well (G1), moderately (G2), or poorly (G3) differentiated hepatocellular carcinomas (HCCs). A close correlation between the morphological dedifferentiation and increasing copy numbers and variation of FISH signals was seen for chromosomes 1 and 8, respectively (P ≤ 0.0002). Four HCC G1 had constant chromosome patterns for chromosomes 1 and/or 8 with a mean of signals per nucleus ≤5.08 and ≤3 different signal combinations, indicating a low level of CIN, as confirmed by FISH using probes for centromeres of chromosomes 3, 7, and 17. In contrast to this, five HCC G2–3 revealed ≥8.46 signals per nucleus and 23–41 different signal combinations, indicating high levels of CIN. In the remaining cases, signal counts from 5.96–8.46 and 7–15 combinations were seen. Here, nuclei with constant aberration patterns and low copy numbers occurred alongside nuclei with inconstant patterns and high copy numbers. It is evident that in these cases a transition from well to moderately differentiated HCC developed in parallel to an increase in CIN, possibly induced by a major dysregulation of mitotic control mechanisms. In conclusion, CIN may induce a stepwise increase of aneuploidy in HCC that is mirrored by the morphological dedifferentiation of tumor cells.

Nuclear translocation of survivin in hepatocellular carcinoma: a key to cancer cell growth?

Survivin is a recently described anti-apoptosis protein and regulator of cell division. Its expression and localization in hepatocellular carcinoma (HCC) and in normal liver tissue has not been fully elucidated. We examined the expression of survivin, Fas, proliferating cell nuclear antigen (PCNA), and apoptosis in 47 specimens of hepatocellular carcinoma (HCC) and surrounding nonmalignant hepatic tissues. To further determine the relationship between survivin expression and cell proliferation and apoptosis, we performed double immunostaining for survivin and PCNA TUNEL staining in the same HCC specimens. Positive immunostaining for survivin was present in 35 of 47 (74%) HCCs. Twenty-two of 35 survivin-positive HCCs (63%) showed punctate nuclear staining in HCC cells, and the remaining 13 showed predominant cytoplasmic staining. In contrast, nonmalignant hepatocytes showed only cytoplasmic staining. HCC cells had significantly higher PCNA-labeling and apoptotic indices compared with the case of nonmalignant hepatic tissue ( P < 0.001). Furthermore, nucleus-positive HCC specimens for survivin showed the highest PCNA labeling index. The nuclear localization of survivin in HCC cells correlated with tumor cell de-differentiation with the exception of the HepG2 cell line. Survivin expression was inversely associated with apoptosis and was strongly associated with Fas expression ( P = 0.01). All 4 HCC cell lines examined showed survivin expression and punctate nuclear localization. Our results indicate that survivin is localized to the cytoplasm in quiescent nonmalignant liver cells to suppress apoptosis and translocates into the nucleus in HCC cells. In conclusion, translocation of survivin from the cytoplasm to the nucleus may constitute an important regulatory mechanism for cell proliferation and differentiation in HCC.

Prognostic significance of E-cadherin and β-catenin in resected stage I non-small cell lung cancer

E-cadherin and its associated intracellular molecules, catenins, are important for cell-cell adhesion. Impaired expression of these molecules are frequently observed in several cancers. E-cadherin and beta-catenin are often expressed in non-small cell lung cancers. The aim of this study was to investigate the expressions of E-cadherin and beta-catenin and their significance as prognostic markers in pathological stage I non-small cell lung cancer. Paraffin embedded tumor tissue blocks were obtained from 141 patients who underwent resection without preoperative radiotherapy or chemotherapy with pathological stage I non-small cell lung cancer. Tumor samples were prepared in tissue microarrays and they were stained by immunohistochemistry with antibodies against E-cadherin and beta-catenin. The expressions of E-cadherin and beta-catenin were analyzed with relation to the clinico-pathological data. The median follow-up period of the patients was 41 months (range, 2-88 months). Preserved expressions of E-cadherin and beta-catenin were observed in the membrane and the cytoplasm of normal epithelial cells and tumor cells. Absent or reduced expression for E-cadherin and beta-catenin were observed in 60% and 45% of all the patients, respectively. There was a significant positive correlation between E-cadherin and beta-catenin expression (P<0.01). Absent or reduced expression of E-cadherin was observed in 72.5%, 36.6%, and 60.0% of squamous cell carcinoma, adenocarcinoma, and bronchioloalveolar carcinoma, respectively. There was a significant decrease of E-cadherin expression in squamous cell carcinoma compared to adenocarcinoma (P<0.01). Patients with reduced expression of beta-catenin had poor recurrence free survival in adenocarcinoma, but not in squamous cell carcinoma. Decreased expressions of E-cadherin and beta-catenin were closely correlated in resected stage I non-small cell lung cancer. Reduced expression of E-cadherin and beta-catenin indicates tumor cell dedifferentiation and reduced expression of beta-catenin had poor recurrence free survival in adenocarcinoma of the resected stage I non-small cell lung cancer.

Increased frequency of gastrin-releasing peptide receptor gene mutations during colon-adenocarcinoma progression.

Epithelial cells lining the mature human colon do not normally express receptors for gastrin-releasing peptide (GRPR). In contrast, we have shown that when aberrantly expressed in functional form in colon cancer, this protein acted as a morphogen where it caused tumor cells to adopt a better-differentiated phenotype. Importantly, GRPR mRNA is ubiquitously mutated in human colon cancer cell lines, with inactivating mutations detected in all cell lines not expressing functional receptor. Since colon cancers are heterogeneously differentiated, we set out to determine if the GRPR gene was mutated as a function of tumor cell differentiation in archived human colon cancers. We used laser capture microscopy to dissect out 67 regions of defined differentiation from 20 human colon cancers randomly selected from the UIC GI Tumor Bank. Except for two polymorphisms, the GRPR gene was not mutated in nonmalignant epithelial cells. In contrast, 42 distinct mutations were identified in malignant cells. Overall mutation number inversely correlated with the degree of tumor cell differentiation. Within any cancer, all GRPR mutations found within better-differentiated cells were conserved in more poorly-differentiated cells; while all poorly-differentiated cells contained mutations resulting in GRPR pharmacological inactivation. These data suggest that accumulation of mutations within the GRPR gene ultimately resulting in the production of nonfunctional receptors may represent a previously unappreciated mechanism allowing for the dedifferentiation of tumor cells within any particular colon cancer; and that poorly-differentiated tumor cells within any individual cancer may arise clonally from their better-differentiated precursors.

Relationship of the Microvascular Type to the Tumor Size, Arterialization and Dedifferentiation of Human Hepatocellular Carcinoma

Unlike normal liver with the sinusoids, hepatocellular carcinomas (HCCs) possess capillaries. Whether these capillaries derive from the sinusoids remains unclear in human HCCs. This study aimed to examine sinusoidal capillarization in human HCCs and its relationship to the tumor size, arterialization and dedifferentiation. Thirty‐eight HCCs with a diameter of 10–140 mm were pathologically and angiographically examined. By electron microscopy, the microvasculature of tumors was classified into sinusoidal, intermediate and capillary types, which were all negative, partially positive and all positive, respectively, for four parameters, i.e., endothelial defenestration, continuous basement membrane, lack of Kupffer cells, and lack of lipid‐containing hepatic stellate cells. Well‐, moderately and poorly differentiated HCCs displayed sinusoidal/intermediate/capillary types, intermediate/capillary types and only capillary type, respectively, suggesting the transition from the sinusoids to capillaries in well‐differentiated (and probably moderately differentiated) HCCs. Furthermore, well‐differentiated HCCs with a diameter of less than 30 mm often received preferential portal venous blood, while moderately and poorly differentiated ones were all supplied with arterial blood, indicating a relationship between dedifferentiation and arterialization. In contrast, the microvascular type displayed no significant relationship with tumor size or arterialization in well‐differentiated HCCs. The present study has demonstrated that sinusoidal capillarization occurs in human well‐differentiated HCCs and seems to be related to dedifferentiation of parenchymal tumor cells, but not to tumor size or arterialization.

P53 and p21WAF1Expression in Lymphocytic Thyroiditis and Thyroid Tumors

To clarify the roles of increased apoptosis and cell proliferation in chronic autoimmune lymphocytic thyroiditis and thyroid tumorigenesis, expression of p53 and p21WAF1proteins was immunohistochemically investigated in a series of 158 cases. Positive epithelial cells were quantified to give numbers per unit square and to score for distribution. They were found scattered in nontumorous thyroid tissue, their numbers increasing with the severity of thyroiditis and the correlation between expression of the two proteins, regardless of the presence or absence of thyroid neoplasms. Simultaneous expression of both proteins was occasionally found in the same cells by analysis of serial histologic sections. In thyroid tumors, increased expression was found to be diffuse, focal, or scattered for the distribution of p53- or p21WAF1-immunopositive cells in accordance with tumor cell dedifferentiation, showing significant correlation between expression of the two proteins. Correlated with these findings, enhanced apoptosis along with decreased Bcl-2 expression and increased Ki-67 labeling in lymphocytic thyroiditis and thyroid tumors was also confirmed in the same series, usingin situDNA nick-end labeling and immunohistochemical methods. Increased expression of p53 and/or p21WAF1proteins was thus suggestive of possible DNA damage and increased apoptosis in autoimmune thyroiditis. In addition, a significant correlation between protein overexpression and dedifferentiation of thyroid tumor cells was apparent.

Anti-invasion drugs.

Most of the pharmaceuticals in clinical practice today for treatment of breast and other cancers are cytotoxic or cytostatic inhibitors of tumor growth. While this type of drug has found its place, along with surgery and radiotherapy, in treatment of disease, the breast cancer death rate has not decreased. This appears to be the result of rising incidence, resistance to therapy, and metastasis of the disease. Since distant metastasis (usually indicated by lymph node involvement) of breast cancer is related only indirectly to tumor size, it would appear that a concerted effort should be made to discover drugs which directly interfere with this complex process. Metastasis appears to depend upon tumor cell motility, dedifferentiation, local invasion, and angiogenesis. Significant progress has been recently made in the creation of new animal models of metastasis and in identifying several new drugs which may be suitable for clinical inhibition of this process. This article reviews current findings on anti-invasion/metastasis drugs with a focus on breast cancer.

Differences in expression and intracellular distribution of hexokinase isoenzymes in rat liver cells of different transformation stages

The activity, intracellular distribution and mRNA expression of hexokinase isoenzymes were studied in normal rat liver, and in epithelial liver cells at different stages of neoplastic transformation, including non-tumorigenic and tumorigenic cell lines. In contrast to liver, all transformed cells exhibited only hexokinase I and II, which both showed significantly increased activity, hexokinase II being the more abundant form. In parallel, the mRNA expression of the two isoenzymes was elevated, indicating transcriptional control of gene expression. Hexokinase I and II were found in the cytosol and bound to mitochondrial membranes; the percentage of membrane-bound enzyme activity increased with the grade of transformation from 32% of total activity in normal liver up to 69% in dedifferentiated tumor cells. The ratio of hexokinase I/II was higher in the membrane fraction than in the cytosol. In all tissues studied hexokinase II could be resolved in two subtypes IIa and IIb by hydrophobic interaction chromatography. The relative proportion of cytosolic IIa and IIb varied significantly between normal liver (1:1) and transformed cells, and among cells of different transformation stages (4:1 to 1:10). IIa demonstrated the main activity in the more differentiated, IIb in the less differentiated cell lines. IIa-activity showed a good correlation with the intracellular glucose 6-phosphate concentration of the cells. The data indicate that neoplastic cell transformation is accompanied by progressive alterations in the proportion and subcellular distribution of hexokinase isoenzymes I and II.

NON-TELEOLOGICAL DIFFERENTIATION OF MALIGNANCY (REVIEW)

So-called 'de-differentiation' of malignant tumors is not identical with 'retro-differentiation' of normal cells, although induction of differentiation of tumor cells seems to be successful. De-differentiation of tumor cells is a more stochastic process than the reversion of serial developmental lineage in normal differentiation. After retro-differentiation, re-differentiation of normal cells includes serial expression of negative regulators programmed a priori, and a teleological explanation of differentiation is possible. However, 'fragmented' gene expression is generally observed in tumor-cell differentiation. In tumor cells, it is more important that they cannot maintain a differentiated state under normal microenvironments than that they can be induced to differentiate by artificial agents. Induction of differentiation of tumor cells should be different from that of their normal counterparts since loss of regulatory mechanisms could not be restored by the inducers. Phenomenological homology between tumor cells and their normal counterparts in the differentiation may be only an experimental artifact, since the genetic information of development is generally damaged or lost in tumor cells. Loss of teleology in tumor cells is essentially correlated with their selfish behavior. Differentiation is not necessary for tumor cells, since they are 'selfish cells' in the altruistic somatic cell society. The differentiation program of tumor cells should be considered as a remnant of the normal one with loss of teleology.

Breast carcinoma: a collective disorder

The development and differentiation of the epithelial component of glandular tissues such as the breast is regulated by two apparently unrelated processes. One of these is presumed to be epithelial cell collective autonomous, that is, it is mediated by gene products which act directly on the epithelial cells. An important component of autonomous regulation is the functional expression of homotypic cell-cell adhesion molecules such as cadherins. The second process is non-autonomous and involves an inductive effect of the neighboring mesenchymal cell collective. An important component of non-autonomous regulation is the aggregation/condensation of mesenchyme closely associated with the epithelium. We propose that molecular alterations in autonomous and non-autonomous pathways are important causes and indicators respectively of breast cancer progression and that these two fundamental regulators of epithelial collective organization are in fact inter-dependent. For example, we show that the expression of hepatocyte growth factor (HGF), an epithelially targeted mesenchymally derived morphogenic factor is regulated by mesenchymal cell density (condensation) and by factors released from epithelial cells. Breast epithelial cells produce factors which inhibit and stimulate HGF expression. The inhibitory factor is transforming growth factor beta (TGF-beta) and the activation state of TGF-beta is a crucial element in HGF homeostasis. The balance of negative and positive HGF regulators is markedly affected by the growth conditions and differentiation state of the epithelial cells. The expression of the HGF receptor, met, is high in normal breast epithelial cells and in dedifferentiated (ER negative) tumor cells but is reduced or lost in ER positive well differentiated epithelial cells. Our results indicate that the expression of at least one epithelial morphogen, HGF, is inter-dependently regulated by mesenchymal condensation and by factors released by neighboring epithelial cells.

Radioresponsiveness of human glioma, sarcoma, and breast cancer spheroids depends on tumor differentiation

Purpose : Differences in the intrinsic radiosensitivity within and between different tumor classes have been noticed for human tumor cell lines using the clonogenic assay. By far, most of the cell lines studied up to now were derived from poorly differentiated tumors. In this study, the influence of tumor differentiation on the radiation doses necessary to control 50% of small oxic spheroids (SCD 50) was determined. Evidence of a distinct dependence of radioresponsiveness on tumor progression provides a background for an investigation of the underlying mechanisms. Methods and Materials : Spheroids were aggregated from 1000–1500 cells in agarose coated 24 multi-well plates. Their diameters ranged from 156 to 405 μm, depending on the cell line. Spheroids were irradiated with graded 60Co single doses using spheroid control as end point and a minimum follow-up period of 3 months. Results : Cell lines from three low grade gliomas and 10 malignant gliomas were studied in the spheroid control assay. The group mean SCD 50 values were 6.1 ± 1.6 Gy and 13.1 ± 3.3 Gy, respectively. Four cell lines from grade 2 soft tissue sarcomas had a mean SCD 50 value of 6.2 ± 0.5 Gy and one undifferentiated sarcoma line of 11.0 Gy. Three well-differentiated breast cancer lines expressed the cell adhesion molecule E-cadherin, had an epitheloid morphology in monolayer culture, were estrogen receptor positive or contact inhibited in multicellular spheroids. Two undifferentiated breast cancer lines had a fibroblastoid morphology and were marker negative. The mean SCD 50 value of the former was 10.5 ± 1.0 Gy while that of the undifferentiated lines was 14.8 ± 2.8 Gy. Analysis of variance revealed a significant effect of the tumor type as well as the grade of dedifferentiation on the SCD 50 after irradiation with one fraction or 2Gy/fraction. The surviving fractions at 2 Gy (SF2), obtained from the spheroid control rates after different fractionation schedules by approximation of the linear quadratic model assuming Poisson statistics were significantly dependent on tumor type ( p = 0.001, ANOVA F-test) but not on tumor differentiation ( p = 0.27). The αβ ratios did not depend on tumor type ( p = 0.08, ANOVA F-Test) but significantly increased with the grade of tumor cell dedifferentiation ( p = 0.03). Conclusion : The spheroid model is suitable for measuring the radioresponsiveness of differentiated cell lines with very low colony forming efficiencies. Tumor cell differentiation is an important factor for the radioresponsiveness and recovery capacity of human tumor cells.

Simple epithelial cytokeratin-expression in seborrheic keratosis.

The cytokeratin expression of seborrheic keratosis was studied by means of immunohistochemistry and compared with that of normal human skin. The following findings were obtained in seborrheic keratosis: (1) a partial lack of high molecular weight cytokeratin (#1/68 kD, #10/56.6 kD) in all ten cases examined; (2) the detection of cytokeratin typical for simple epithelia (#8/52.5 kD, #18/45 kD, #19/40 kD) in eight of ten cases; and (3) the detection of cytokeratin #5/58 kD in suprabasal cells in 5 of 10 cases. An immunoelectron-microscopic investigation, using an anti-keratin antibody against cytokeratin #19/40 kD, revealed a whirl-like arrangement of keratin filaments within immunoreactive cells, in contrast to a linear, parallel arrangement in non-immunoreactive cells. Cells known to express cytokeratin typical for simple epithelia, such as sweat gland cells or Merkel cells, were not observed. The altered cytokeratin gene-expression in seborrheic keratosis may be attributable to de-differentiation of tumor cells or potential re-differentiation towards embryonic keratinocytes.