Comparison of morphonuclear features in normal, benign and neoplastic thyroid tissue by digital cell image analysis.

We analyzed the morphonuclear features of thyroid cell nuclei from 10 normal tissues (10 multinodular goiters), 10 benign tissues (10 adenomas) and 10 neoplastic tissues (10 carcinomas--2 follicular, 2 medullary and 6 papillary). These morphonuclear features quantitatively described the nuclear size, DNA content and chromatin texture of 200-400 Feulgen-stained cell nuclei that were digitized by means of a cell image processor. Nuclear DNA estimations revealed that the benign thyroid tumors showed an aneuploidy level that was not different from that of the neoplastic tissues. Morphometric measurements showed a significant and positive correlation between an increase in nuclear area and the development of thyroid pathology--i.e., from normal to a neoplastic stage. We also observed a significant decrease in chromatin condensation and heterogeneity from normal to neoplastic thyroid tissue. In the specific case of thyroid tumors, such criteria were not found sufficient per se for a diagnosis of malignancy. More detailed data banks will be necessary for this purpose.     

Influence of smear preparation and fixatives on the DNA ploidy and the morphonuclear features of the MXT mammary tumor and normal tissues in the mouse

We compared cytomorphonuclear parameters--related to DNA histogram and chromatin distribution--on MXT mouse mammary tumor and murine normal cells from fresh squash smears or from deparaffinized tissue smears fixed in several fluids. We used the SAMBA 200 cell image processor with software allowing for the discrimination of parameters computed on Feulgen-stained nuclei. The spectrophotometric results--assessed by integrated optical density values--indicate that the nuclei from deparaffinized tissue fixed in Bouin's fluid are around 50% less stained than those fixed in formalin or ethanol-formalin-acetic acid (EFA). The fresh smears of nuclei fixed in formalin contain a less well-defined and more homogeneous chromatin than after Bouin's or EFA fixation. This has led to the conclusion that morphonuclear parameter comparisons performed on tissues differently processed or from different origins present severe limitations.     

Initiation of lymphocyte DNA synthesis

The initiation of DNA replication in T lymphocytes appears to be regulated by two distinct activities: one associated with proliferation which mediates initiation, and another associated with quiescence which blocks initiation. Activated lymphocytes and proliferating lymphoid cell lines produce an activity, termed ADR, which can initiate DNA replication in isolated, quiescent nuclei. ADR is heat-labile, has protease activity or interacts closely with a protease, and is distinct from the DNA polymerases. ADR activity is absent in quiescent lymphocytes and appears in mitogen-stimulated lymphocytes after IL-2 binding. The generation of active ADR appears to be mediated by phosphorylation of a precursor which is present in resting cells. Nuclei from mitogen-unresponsive lymphocytes fail to initiate DNA replication in response to ADR, of potential importance in the age-related decline of immunity. Quiescent lymphocytes lack ADR and synthesize an ADR-inhibitory activity. The ADR inhibitor is a heat-stable protein which suppresses the initiation of DNA synthesis, but is ineffective at suppressing elongation once DNA strand replication has begun. Nuclei from several neoplastic cell lines fail to respond to the ADR inhibitor, which may play a role in the continuous proliferation of these cells. At least one of these neoplastic cell lines produces both ADR and an inhibitory factor. These findings suggest that the regulation of proliferation is dependent on the balance between activating and inhibitory pathways.     

Mechanisms of cross-resistance between nucleoside analogues and vincristine or daunorubicin in leukemic cells

The aim of this study was to clarify the biochemical and molecular mechanisms behind the cross-resistance to nucleoside analogues (NAs) in four erythroleukemic cell lines with acquired resistance to the anthracycline daunorubicin and to the vinca alkaloid vincristine, expressing high levels of p-glycoprotein (P-gp, MDR1). All resistant strains exhibited cross-resistance to NA (cladribine and cytosine arabinoside)-induced apoptosis, assessed by caspase-3-like activation and were less sensitive to NA cytotoxicity in MTT assay. Real-time PCR and enzyme activity analysis showed reduced amounts of deoxycytidine kinase (35-80%) and elevated levels of 5'-nucleotidases (50-100%). The ratio 5'-nucleotidase to deoxycytidine kinase increased between 2.5- and 7.5-folds in resistant cells. This is in agreement with the observation that 5'-nucleotidase/deoxycytidine kinase ratio might be an important factor in predicting resistance to NAs. Implications of this finding for combining anthracyclines or vinca alkaloids with NAs toward leukemic cells are discussed.     

Studies on the biology,histology and cytogenetics of two sublines of a murine ovarian reticular cell sarcoma

Two sublines of the ovarian reticular cell sarcoma M5 in C57BL mice respond differently to cyclophosphamide and other alkylating agents. The subline R16, which is resistant to cyclophosphamide, was obtained by treating M5 mice repeatedly with this compound and subsequently transplanting the regrowing tumor for 16 passages. The R16 subline shows biological characteristics perfectly superimposable to those of the parent line and histologically resembles an undifferentiated mesenchymal neoplasia with numerous atypical nuclei and karyokinetic figures with large necrotic areas. The cytogenetic examination of the distribution in the chromosomal number of R16 indicates that this subline may be considered a clone of the parent line with a modal class of 35 chromosomes (34–37) versus a class of 34 (31–37) in the M5 tumor line. The presence of metacentric chromosomes characterizes the modal class of the two lines, 23 in the R16, and 25 in the M5 tumor lines.Abbreviations M5 murine ovarian reticular cell sarcoma - M5-CTX-16R(R16) subline of M5 made resistant to cyclophosphamide - CTX cyclophosphamide     

Isolation and partial characterization of human cell mutants differing in sensitivity to killing and mutation by methylnitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine

Isogenic variants resistant to alkylating agents have been isolated from the human lymphoblast cell line TK6. The cell lines may be divided into four classes on the basis of resistance to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The sensitive TK6 parental line shows a 37% survival after 45-min exposure to 0.04 microM MNNG; the three classes of more resistant mutants show 37% survival after 45-min exposure to 2 microM (MF lines), 6 microM (MT lines), and greater than or equal to 10 microM (MX line) MNNG. A representative MF line, MF1, is resistant to both killing and mutation by MNNG or N-methyl-N-nitrosourea. An MT clone, MT1, is highly resistant to killing but hypermutable by MNNG. The MT1 line, like the parental TK6, does not remove O6-methylguanine adducts from the DNA. Our data are consistent with the hypothesis that the MT1 line possesses a nonexcision pathway of defense against killing by alkylating agents. Rather than preventing alkylation of DNA or removing alkylated adducts, the MT1 cells appear to be tolerant of the adducts that are not removed from the DNA.     

Identification of the Yc1 glutathione S-Transferase mRNA as the overexpressed species in a nitrogen mustard–resistant rat mammary carcinoma cell line

Glutathione transferases (GSTs) have been shown to be overexpressed in a number of tumor cell lines selected for resistance to chemotherapeutic drugs and have been implicated in some studies of clinical specimens. In tumor cell lines selected for resistance to chemicals that alkylate DNA, the isoform most frequently overexpressed is GST-Yc, a member of the α class GSTs. To date, two variations of the cDNA designated Yc₁ with subtle differences have been described, and Yc₂ is shown to be clearly distinct. Transfection of a Yc₁ cDNA constitutively expressed in rat liver into rat mammary cancer cells confers resistance to alkylators, however, to a lesser extent than is observed in the cells selected for resistance. It has therefore been widely suggested that the GST that is overexpressed in selected resistant cells represents a distinct and novel isoform. We have previously described a rat mammary carcinoma cell line (MLNr) that is resistant to alkylating agents, and overexpresses a GST with characteristics similar to GST-Yc₁ and not Yc₂. It has many features common to the several other GST-Yc overexpressing alkylator resistant cell lines. We have cloned the specific Yc cDNA overexpressed in MLNr and analyzed it in detail and found that it is identical to one of the previously reported Yc₁ cDNAs, suggesting that there is no additional Yc gene specifically induced by nitrogen mustards. Another hypothesis to explain the difference in the level of resistance in selected versus GST-Yc transfected cells is the lack of concurrent increased glutathione (GSH) in the transfectants, which is a common feature in the selected resistant cells. Experiments in which we modulated GSH levels suggest that this is not likely. These studies add to our speculation that other mechanisms may be involved in alkylator resistance. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 12: 11–17, 1998     

Comparison of fine needle aspirates of breast cancers to imprint smears by means of digital cell image analysis

Morphonuclear assessments were performed using the SAMBA 2005 cell image processor on cell nuclei in fine needle aspirates and corresponding imprint smears from 17 not-otherwise-specified (NOS) breast carcinomas to study the influence of cell sampling on the morphonuclear measurements. Fourteen parameters related to densitometric (nuclear DNA content), morphometric (nuclear area) and textural (chromatin organization and distribution) characteristics were computed for each nucleus. The results demonstrated that such morphonuclear features evolved significantly and positively with respect to conventional histopathologic grading. The method of cell sampling significantly influenced the results, but without altering the general conclusions regarding evolution of the morphonuclear features.     

Cytogenesis of murine mammary tumors in BALB/cfC3H and BALB/cfRIII strains

Biological and morphological differences in the mammary tumors of BALB/cfC3H and BALB/cfRIII mice are due to differences in the causative viruses. The C3H and RIII variants of the murine mammary tumor virus (MuMTV) might give origin to different mammary tumors by transforming different types of cell, i.e. epithelial or myoepithelial cells. The nature (epithelial or myoepithelial) of the neoplastic cells has been investigated by demonstrating their plasma membrane ATPase activities. We found that in normal murine mammary gland both epithelium and myoepithelium have Mg++ dependent ATPase activity, while the myoepithelium shows in addition an Na+K+ dependent ATPase activity. It is suggested that the results obtained exclude the participation of myoepithelium to the neoplastic growth and we ascribe the differences in mammary tumors of the two strains of mice to differences in the mechanisms of action of the virus variants.     

Proteome analysis of vinca alkaloid response and resistance in acute lymphoblastic leukemia reveals novel cytoskeletal alterations

Vinca alkaloids are used widely in the treatment of both childhood and adult cancers. Their cellular target is the beta-tubulin subunit of alpha/beta-tubulin heterodimers, and they act to inhibit cell division by disrupting microtubule dynamics. Despite the effectiveness of these agents, drug resistance is a major clinical problem. To identify the underlying mechanisms behind vinca alkaloid resistance, we have performed high resolution differential proteome analysis. Treatment of drug-sensitive human leukemia cells (CCRF-CEM) with vincristine identified numerous proteins involved in the cellular response to vincristine. In addition, differential protein expression was analyzed in leukemia cell lines selected for resistance to vincristine (CEM/VCR R) and vinblastine (CEM/VLB100). This combined proteomic approach identified 10 proteins altered in both vinca alkaloid response and resistance: beta-tubulin, alpha-tubulin, actin, heat shock protein 90beta, 14-3-3tau, 14-3-3epsilon, L-plastin, lamin B1, heterogeneous nuclear ribonuclear protein-F, and heterogeneous nuclear ribonuclear protein-K. Several of these proteins have not previously been associated with drug resistance and are thus novel targets for elucidation of resistance mechanisms. In addition, seven of these proteins are associated with the tubulin and/or actin cytoskeletons. This study provides novel insights into the interrelationship between the microtubule and microfilament systems in vinca alkaloid resistance.     

Transforming potential of alternatively spliced variants of fibroblast growth factor receptor 2 in human mammary epithelial cells

A breast cancer cell line developed in our laboratory (SUM-52PE) has a 12-fold amplification and high-level overexpression of the oncogene fibroblast growth factor receptor 2 (FGFR2). Previously, nine different alternatively spliced FGFR2 variants were isolated from this cell line. Overexpression of two variants that differ only in their carboxyl termini (C1 and C3) has been successfully accomplished in the immortalized human mammary epithelial cell line H16N2. FGFR2 expression led to the activation of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling cascades. Phosphorylation of the adapter protein FGF receptor substrate 2 is much more robust in the cells expressing the C3 variant of FGFR2 compared with the C1 variant. H16N2 cells expressing the full-length FGFR2 with the C1 or C3 carboxyl terminus were tested for their ability to grow under epidermal growth factor (EGF)-independent conditions, in soft agar, and for their ability to invade naturally occurring basement membranes and compared with the parental SUM-52PE cell line. All three cell lines grew under EGF-independent conditions and all were inhibited by the FGFR family specific inhibitor PD173074. The full-length FGFR2-C1 and FGFR2-C3 variants grew robustly in soft agar similar to the parental cell line SUM-52PE. However, cells expressing the C3 variant formed large colonies in agar in both insulin-free and EGF-free medium, whereas the cells expressing the C1 variant required insulin for growth. Soft agar growth was also inhibited by PD173074. Because SUM-52PE was developed from a metastatic breast carcinoma, the FGFR2-overexpressing cell lines were assessed for their ability to invade sea urchin embryo cell membranes. H16N2 cells expressing the C1 carboxyl terminus failed to invade sea urchin embryo cell membranes. By contrast, FGFR2-C3-expressing cells were as invasive as the SUM-52 breast cancer cells and erbB-2-overexpressing H16N2 cells. These results indicate that FGFR2 is a transforming oncogene in human mammary epithelial cells when expressed to levels similar to that found in breast cancer cells with FGFR2 gene amplification. Furthermore, the results suggest that different splice variants have differing transforming activities and that signaling from variants expressing the C3 carboxyl terminus results in more autonomous signaling, cell growth, and invasion.     

Genes that cooperate with tumor promoters in transformation

Tumor-promoting phorbol esters, like growth factors, elicit pleiotropic responses involving biochemical pathways that lead to different biological responses. Genetic variant cell lines that are resistant to mitogenic, differentiation, or transformation responses to tumor promoters have been valuable tools for understanding the molecular bases of these responses. Studies using the mouse epidermal JB6 cell lines that are sensitive or resistant to tumor promoter-induced transformation have yielded new understanding of genetic and signal transduction events involved in neoplastic transformation. The isolation and characterization of cloned mouse promotion sensitivity genes pro-1 and pro-2 is reviewed. A new activity of pro-1 has been identified: when transfected into human cancer prone basal cell nevus syndrome fibroblasts but not normal fibroblasts mouse pro-1 confers lifespan extension on these cells. Recently, we have found that a pro-1 homolog from a library of nasopharyngeal carcinoma, but not the homolog from a normal human library, is activated for transferring promotion sensitivity. The many genetic variants for responses to tumor promoters have also proved valuable for signal transduction studies. JB6 P- cells fail to show the 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced synthesis of two proteins of 15 and 16 kD seen in P+ cells. P-, P+, and TPA transformed cells show a progressive decrease in both basal and TPA-inducible levels of a protein kinase C substrate of 80 kD. P- cells are relatively resistant both to anchorage-independent transformation and to a protein band shift induced by the calcium analog lanthanum. It appears that one or more calcium-binding proteins and one or more pro genes may be critical determinants of tumor promoter-induced neoplastic transformation.     

Fluctuation of simian virus 40 (SV40) super T-antigen expression in tumors induced by SV40-transformed mouse mammary epithelial cells.

Higher-molecular-weight forms of the simian virus 40 (SV40) large tumor antigen (T-Ag), designated super T-Ag, are commonly found in SV40-transformed rodent cells. We examined the potential role of super T-Ag in neoplastic progression by using a series of clonal SV40-transformed mouse mammary epithelial cell lines. We confirmed an association between the presence of super T-Ag and cellular anchorage-independent growth in methylcellulose. However, tumorigenicity in nude mice did not correlate with the expression of super T-Ag. In the tumors that developed in nude mice, super T-Ag expression fluctuated almost randomly. Cell surface iodination showed that super T-Ag molecules were transported to the epithelial cell surface. The biological functions of super T-Ag remain obscure, but it is clear that it is not important for tumorigenicity by SV40-transformed mouse mammary epithelial cells. Super T-Ag may be most important as a marker of genomic rearrangements by the resident viral genes in transformed cells.     

Inhibition of carcinogenesis by retinoids.

Experimental investigations of the antineoplastic effects of retinoids are reviewed in this paper. In vitro studies have shown that the hyperplastic and metaplastic response to chemical carcinogens of mouse prostate cultures is suppressed by the addition of retinoids to the culture medium, that retinoids can partially inhibit the morphologic transformation of 10T 1/2 cells by physical or chemical carcinogens, and that the growth of some non-neoplastic and some neoplastic cell lines can be inhibited by retinoids. In vivo studies have shown that retinoids can suppress papilloma and carcinoma development (the promotion phase) in the two-stage skin carcinogenesis assay, inhibit mammary and bladder carcinogenesis in mice and rats, and inhibit the growth of some transplantabletumor lines. So far it has not been possible to inhibit predictably tumour formation in the intestinal tract or the respiratory tract of rodents. Almost all the synthetic retinoids have a higher therapeutic index than the natural retinoids in the prevention or treatment of cancer.     

Possible role of genetic cell variants in the viral induction of mouse mammary tumors

Out of three attempts to induce neoplasia in normal C57Bl mammary epithelial cells with the mouse mammary tumor virus (MuMTV) only one presented signs of tumorigenicity. Immunofluorescence showed that virus synthesis took place in all three sublines but tumorigenicity as detected by cell aggregation viability (CAV) and transplantation into syngeneic mice failed to occur in two of them. By comparison, cells from a BALB/c spontaneous mammary tumor that do not express MuMTV were 100% tumorigenic, whereas cells from a BALB/cfC3H tumor with a 95% virus-producing cell population had a normal CAV and were tumorigenic only in 60% of the test animals. This lack of correlation suggested that many of the virus-producing cell were not neoplastic and that neoplasia might occur under virus stimulation only if a restricted population of genetic cell variants existed. Accelerated tissue culture passages of virus-free C57Bl and BALB/c normal mammary cells resulted in their spontaneous neoplasia at Passages 23 and 50, respectively; when duplicated cells cryopreserved in early passages were revived and cultivated in the same manner, neoplasia occurred at Passages 27 and 58. The similarity of the passage numbers appears to confirm the existence of genetic cell variants among the normal cell population.     

In vivo relationship between morphonuclear parameters and hormone sensitivity of the murine MXT mammary tumor

The initially hormonosensitive (HS) MXT mouse mammary tumor spontaneously evolved to hormonoin-dependence (HI). Using a SAMBA 200 cell image processor, we compared the DNA content and the chromatin structure of HS and HI tumor cells squashed onto histologic slides; the nuclei were colored by the Feulgen reaction. We compared HI and HS nuclei by a discriminant analysis using the 15 parameters obtained on each nucleus. We show that the percentage of well-classified granulocytes (2n DNA content control) versus HS or HI nuclei exceeded 99. On the other hand, this percentage did not reach 70 when we compared HS and HI. The cell cycle analysis revealed that the percentage of cells in S and G2 + M phases were significantly higher in HI tumors than in the HS. Hence, HI and HS MXT tumor nuclei seem to be morphologically identical, but are significantly different if we refer to cell proliferation rates.     

Characterization of androgen uptake by purified nuclei from an androgen-dependent and two androgen-independent cell lines of the shionogi mouse mammary carcinoma

Properties of androgen uptake by nuclei prepared from an androgen-dependent and two androgenindependent tumour cell lines from the Shionogi mouse mammary carcinoma were characterized. It was found that:1. Maximal uptake of dihydrotestosterone (DHT) by the androgen-dependent tumour cell line occurred after 2 h incubation at 20°C; 2. Nuclei from three tumour cell lines displayed similar affinity for DHT but the two androgen-independent cell lines had less than one-quarter the number of uptake sites; 3. Loss of label from nuclei which had been pre-incubated with [³H]-DHT for 18 h at 20°C was greater from the AD cell line nuclei than from the androgen-independent cell lines; 4. Whole cell contamination of the nuclear preparations did not contribute to specific DHT uptake.     

Mouse Mammary Tumor Virus Suppresses Apoptosis of Mammary Epithelial Cells through ITAM-Mediated Signaling

Many receptors in hematopoietic cells use a common signaling pathway that relies on a highly conserved immunoreceptor tyrosine-based activation motif (ITAM), which signals through Src family tyrosine kinases. ITAM-bearing proteins are also found in many oncogenic viruses, including the mouse mammary tumor virus (MMTV) envelope (Env). We previously showed that MMTV Env expression transformed normal mammary epithelial cells and that Src kinases were important mediators in this transformation. To study how ITAM signaling affects mammary cell transformation, we utilized mammary cell lines expressing two different ITAM-containing proteins, one encoding a MMTV provirus and the other a B cell receptor fusion protein. ITAM-expressing cells were resistant to both serum starvation- and chemotherapeutic drug-induced apoptosis, whereas cells transduced with these molecules bearing ITAM mutations were indistinguishable from untransduced cells in their sensitivity to these treatments. We also found that Src kinase was activated in the MMTV-expressing cells and that MMTV-induced apoptosis resistance was completely restored by the Src inhibitor PP2. In vivo, MMTV infection delayed involution-induced apoptosis in the mouse mammary gland. Our results show that MMTV suppresses apoptosis through ITAM-mediated Src tyrosine kinase signaling. These studies could lead to the development of effective treatment of nonhematopoietic cell cancers in which ITAM-mediated signaling plays a role.     

The isolation and genetic analysis of V79-derived etoposide sensitive Chinese hamster cell mutants: two new complementation groups of etoposide sensitive mutants

Using a replica plating microwell method, three Chinese hamster V79-derived cell lines, designated ETO1, ETO2 and ETO3, which exhibit hypersensitivity to the non-intercalating topoisomerase II inhibitor etoposide have been isolated. Mutant lines ETO2 and ETO3 are cross-sensitive to the topoisomerase II inhibitors adriamycin and streptonigrin; however, neither mutant is sensitive to the topoisomerase I inhibitor camptothecin, the bifunctional alkylating agent mitomycin C, nor hydrogen peroxide. In contrast, ETO1 is cross-sensitive to camptothecin but displays only slight sensitivity to adriamycin, streptonigrin and hydrogen peroxide, and is not sensitive to mitomycin C. It has been established through extensive cell fusion studies that all three mutants are genetically distinct, and that ETO2 and ETO3 genetically complement all other known etoposide-sensitive Chinese hamster cell mutants (i.e., irs1, XR-1, xrs1, V3, BLM2, ADR1, ADR3, ADR4 and ADR5) thus defining two new complementation groups of etoposide sensitive mutants. Interestingly, the hybrids created by the fusion irs2TOR (thioguanine and ouabain resistant)xETO1 and the reciprocal cross ETO1TORxirs2 both exhibited a response to camptothecin intermediate with respect to V79 and ETO1. It has been hypothesised that this partial complementation may be the result of intragenic complementation and that both ETO1 and irs2 result from mutations in the gene XRCC8. This study indicates that cellular responses to topoisomerase II inhibitors are complex and hypersensitivity may result from mutations in many different genes.