M. Balzi

Cell Cycle-dependent Expression of HERG1 and HERG1B Isoforms in Tumor Cells

The role of K+ channel activity during cell cycle progression has become a research topic of considerable interest. Blocking of K+ channels inhibits the proliferation of many cell types, although the mechanism of this inhibition is unclear. There is speculation that K+channels differentially regulate the electrical potential of the plasma membrane (V m ) during proliferation. We have demonstrated that in tumor cells the value of V m is clamped to rather depolarized values by K+ channels belonging to the HERG family. We report here that tumor cell lines preferentially express the herg1 gene and a truncated,N-deleted form that corresponds to herg1b. This alternative transcript is also expressed in human primary acute myeloid leukemias. Both HERG1 and HERG1B proteins are expressed on the plasma membrane of tumor cells and can form heterotetramers. The expression of HERG protein isoforms is strongly cell cycle-dependent, accounting for variations in HERG currents along the mitotic cycle. Moreover, the blocking of HERG channels dramatically impairs cell growth of HERG-bearing tumor cells. These results suggest that modulated expression of different K+ channels is the molecular basis of a novel mechanism regulating neoplastic cell proliferation.

HERG potassium channels are constitutively expressed in primary human acute myeloid leukemias and regulate cell proliferation of normal and leukemic hemopoietic progenitors.

An important target in the understanding of the pathogenesis of acute myeloid leukemias (AML) relies on deciphering the molecular features of normal and leukemic hemopoietic progenitors. In particular, the analysis of the mechanisms involved in the regulation of cell proliferation is decisive for the establishment of new targeted therapies. To gain further insight into this topic we report herein a novel approach by analyzing the role of HERG K+ channels in the regulation of hemopoietic cell proliferation. These channels, encoded by the human ether-a-gò-gò-related gene (herg), belong to a family of K+ channels, whose role in oncogenesis has been recently demonstrated. We report here that herg is switched off in normal peripheral blood mononuclear cells (PBMNC) as well as in circulating CD34+ cells, however, it is rapidly turned on in the latter upon induction of the mitotic cycle. Moreover, hergappears to be constitutively activated in leukemic cell lines as well as in the majority of circulating blasts from primary AML. Evidence is also provided that HERG channel activity regulates cell proliferation in stimulated CD34+ as well as in blast cells from AML patients. These results open new perspectives on the pathogenetic role of HERG K+ channels in leukemias.

Evidence for reciprocity of bcl-2 and p53 expression in human colorectal adenomas and carcinomas

Evidence of accumulating for the failure of apoptosis as an important factor in the evolution of colorectal cancer and its poor response to adjuvant therapy. The proto-oncogene bcl-2 suppresses apoptosis. Its expression could provide an important survival advantage permitting the development of colorectal cancer. The expression of bcl-2 and p53 was determined by immunohistochemistry in 47 samples of histologically normal colonic mucosa, 19 adenomas and 53 adenocarcinomas. Expression of bcl-2 in colonic crypts > 5 cm from the tumours was confined to crypt bases but was more extensive and intense in normal crypts < 5 mm from cancers. A higher proportion of adenomas (63.2%) than carcinomas (36.5%) expressed bcl-2 (P < 0.05). A lower proportion of adenomas (31.6%) than carcinomas (62.3%) expressed p53 (P < 0.02). A total of 26.3% of adenomas and 22% of carcinomas expressed both bcl-2 and p53. To determine whether these samples contained cells which expressed both proteins, a dual staining technique for bcl-2 and p53 was used. Only 1/19 adenomas and 2/53 carcinomas contained cells immunopositive for both bcl-2 and p53. Moreover there was evidence of reciprocity of expression of bcl-2 and p53 in these three double staining neoplasms. We suggest that bcl-2 provides a survival advantage in the proliferative compartment of normal crypts and colorectal neoplasms. However, its expression is lost during the evolution from adenoma to carcinoma, whereas p53 expression is increased, an event generally coincident with the expression of stabilised p53, which we presume to represent the mutant form.

Bcl-w expression in colorectal adenocarcinoma.

We have found that the anti-apoptotic Bcl-2 family protein, Bcl-w, was frequently expressed in colorectal adenocarcinomas, with 69/75 showing positive staining with anti-Bcl-w IgG. Adenomas demonstrated a much lower frequency of Bcl-w expression (only 1 of 17), as did adenocarcinomas from other epithelial tissues such as breast (0/8), stomach (1/12) and cervix (0/12). Bcl-w status could be related to the histopathological classification of the tumours, with TNM stage III tumours showing significantly higher levels of expression than tumours of better prognostic grade (at P = 0.009). Those patients with node involvement also had tumours with significantly elevated levels of Bcl-w (at P = 0.02), compared to those which were node-negative. The results suggest that Bcl-w could play a general role in the progression from adenoma to adenocarcinoma in the colorectal epithelium. Currently, more data are being collected to allow us to assess the importance of Bcl-w for disease progression and patient survival.

Prognostic significance of biologic markers in node-negative breast cancer patients : a prospective study

It is generally thought that future advances in the treatment and cure of breast cancer patients will be made possible through a deeper understanding of tumor biology and an improved capability to define the prognosis of each single patient. This will lead to the formulation of new, more selective, and patient-tailored therapies. It is therefore important, when studying potential prognostic factors, to follow methodologic requirements and guidelines which involve the carrying out of prospective studies as confirmatory steps. Repeatedly or recently investigated prognostic markers (tumor size, menopausal status, ER, PgR, 3H thymidine labeling index, c-erbB-2 and p27 expression) were evaluated on a series of 286 prospectively recruited node negative breast cancer patients who underwent loco-regional treatment alone and were closely followed. The individual and relative prognostic contribution of each variable with respect to other factors, as well as their ability to identify node negative patients at risk, were assessed by univariate and multivariate analysis. At a five-year follow-up, only tumor size (p = 0.021) and TLI (p = 0.016) individually proved to be significant prognostic indicators of relapse-free survival. Conversely, p27 expression was not related to RFS and c-erbB-2 expression appeared to have only a short-term effect on patient prognosis. TLI and tumor size, tested in multivariate analysis along with ER and menopausal status, maintained their independent prognostic relevance. The study, performed on a large series of node-negative patients given loco-regional treatment alone, for the first time prospectively recruited, showed the prognostic relevance of TLI and its independence from other clinico-pathologic and biologic factors over a five-year period.

Carborane derivatives loaded into liposomes as efficient delivery systems for boron neutron capture therapy.

Boron neutron capture therapy (BNCT) is an anticancer therapy based on the incorporation of (10)B in tumors, followed by neutron irradiation. Recently, the synthesis and delivery of new boronated compounds have been recognized as some of the main challenges in BNCT application. Here, we report on the use of liposomes as carriers for BNCT active compounds. Two carborane derivatives, i.e., o-closocarboranyl beta-lactoside (LCOB) and 1-methyl-o-closocarboranyl-2-hexylthioporphyrazine (H(2)PzCOB), were loaded into liposomes bearing different surface charges. The efficacy of these formulations was tested on model cell cultures, that is, DHD/K12/TRb rat colon carcinoma and B16-F10 murine melanoma. These induce liver and lung metastases, respectively, and are used to study the uptake of standard BNCT drugs, including borophenylalanine (BPA). Boron concentration in treated cells was measured by alpha spectrometry at the TRIGA mark II reactor (University of Pavia). Results showed high performance of the proposed formulations. In particular, the use of cationic liposomes increased the cellular concentration of (10)B by at least 30 times more than that achieved by BPA.

The effects of irradiation at different times of the day on rat intestinal goblet cells

Quantitative changes in jejunal goblet cells were studied in control and whole body irradiated rats using PAS‐Alcian blue staining of crypt sections. A circadian dependence was observed when control animals were killed at different times during the light/dark cycle. Irradiation with 3 Gy produced a 2–3‐fold increase within 36 h in goblet cells relative to controls, followed by a reduction to very low levels. There was a return to pre‐treatment levels later than was observed for the columnar cells. The present results on the pattern of response of goblet cells and those of brush border enzyme activity are consistent with the hypothesis that ionizing radiation can influence differentiation. In fact during the first hours after irradiation an early induction of differentiation is evident while during the early repopulation phase columnar cells prevailed relative to the goblet cells. Only at later times were normal differentiation patterns seen. Groups of animals exposed to the same dose of radiation at different times of the day showed similar general patterns of behaviour even if the group irradiated at midnight showed a more marked and longer lasting injury.

Relationship between cytosol TPS, TPA and cell proliferation

The serological tumor marker tissue polypeptide antigen (TPA) and the more recently identified tissue-specific polypeptide antigen (TPS) have been reported to be indicators of the proliferation rate of the tumor. In the present investigation we compared the cytosol level of the two markers with the proliferative activity of the tumor measured using the 3H-thymidine labelling index. The preliminary results presented here show that higher TLI is associated with lower cytosol levels of both TPA and TPS. TPA and TPS in the cytosol were significantly associated. These findings are in agreement with the previously demonstrated association between high TPA cytosol levels and better prognosis in breast cancer. Further studies are ongoing in order to: 1. confirm these findings in a larger patient series; 2. investigate any possible prognostic indication provided by TPS; 3. evaluate any possible biological meaning of the negative association between TPA/ TPS and TLI in the cytosol of breast cancer.

Irradiation at Different Times of the Day Morphology and Kinetics of the Small Intestine

Rats were irradiated at different time of the day with sublethal doses on the abdomen only, and qualitative and quantitative morphologic modifications were determined. The experiments seemed to be demonstrate that in the groups irradiated at night and at the end of the light period early injury is not severe whereas in the group irradiated at the end of the dark period repair of the injury seems to be more effective.

In vivo effects of rosiglitazone in a human neuroblastoma xenograft

Background:Neuroblastoma (NB) is the most common extra-cranial solid tumour in infants. Unfortunately, most children present with advanced disease and have a poor prognosis. There is in vitro evidence that the peroxisome proliferator-activated receptor γ (PPARγ) might be a target for pharmacological intervention in NB. We have previously demonstrated that the PPARγ agonist rosiglitazone (RGZ) exerts strong anti-tumoural effects in the human NB cell line, SK-N-AS. The aim of this study was to evaluate whether RGZ maintains its anti-tumoural effects against SK-N-AS NB cells in vivo.Methods and results:For this purpose, tumour cells were subcutaneously implanted in nude mice, and RGZ (150 mg kg−1) was administered by gavage daily for 4 weeks. At the end of treatment, a significant tumour weight inhibition (70%) was observed in RGZ-treated mice compared with control mice. The inhibition of tumour growth was supported by a strong anti-angiogenic activity, as assessed by CD-31 immunostaining in tumour samples. The number of apoptotic cells, as determined by cleaved caspase-3 immunostaining, seemed lower in RGZ-treated animals at the end of the treatment period than in control mice, likely because of the large tumour size observed in the latter group.Conclusions:To our knowledge, this is the first demonstration that RGZ effectively inhibits tumour growth in a human NB xenograft and our results suggest that PPARγ agonists may have a role in anti-tumoural strategies against NB.