Liposomal Targeting of Bcl-2 Antisense Oligonucleotides with Enhanced Stability into Human Myeloma Cell Lines

Cationic liposomes improve the delivery of antisense oligonucleotides (ODNs) into cells. However, there is marked variability in the cellular uptake of ODNs into different cell lines. We used liposomes containing dimethyloctadecylammonium bromide (DDAB) and dioleoylphosphatidylethanolamine (DOPE) to increase the delivery of phosphodiester ODNs into four different myeloma cell lines. The delivery by cationic liposomes increased the delivery of bcl-2 antisense ODNs by a factor of 9 to 45 as compared to plain ODNs. The stability of ODNs was increased with liposomes both in the culture medium and within the cells. Intact liposomal ODNs were detected inside the cells up to 24 hours with gel electrophoresis and phosphor imager analysis. Antisense ODNs had no effect on bcl-2 mRNA levels. Also the proliferation of myeloma cells remained unchanged during the 3-day incubation period. Our study shows that liposomal antisense ODNs targeting bcl-2 of human myeloma cells result in increased stability of ODNs with minimal toxicity. However, further modifications are needed to gain biological effects of antisense ODNs on human myeloma cells.     

Squamous cell carcinoma of the conjunctiva. Failure to demonstrate HPV DNA by in situ hybridization and polymerase chain reaction.

Squamous cell carcinoma of the conjunctiva is a distinct rarity, often arising at the corneoscleral limbus and initially resembling pterygium or chronic keratoconjunctivitis. In this paper we report 4 patients with conjunctival squamous cell carcinoma/carcinoma in situ, which comprise all the cases found in the files of Kuopio University Hospital during 1959-1991. The clinical appearance, diagnosis and treatment of the lesions are described. All biopsies were studied for the presence of Human papillomavirus (HPV) DNA (recently demonstrated in conjunctival squamous cell papillomas, precancer lesions and carcinomas) by using in situ DNA hybridization (ISH) and polymerase chain reaction (PCR). Both techniques failed to demonstrate the DNA of any of the following HPV types: HPV 6, 11, 16 and 18 in any of the lesions. The results are discussed in the light of the recently proposed HPV etiology of these lesions.     

Nucleolar organiser regions (AgNORs) related to histopathological characteristics and survival in prostatic adenocarcinoma.

The number of silver stained nucleolar organiser regions (AgNORs) was assessed in biopsy specimens of 78 patients with prostatic adenocarcinoma followed up for a mean of 15.6 years. The number of Ag-NORs was related to histological features, clinical stage, DNA ploidy, S-phase fraction (SPF) and clinical outcome. In 31/36 (86%) of grade I tumours on average less than 3.5 AgNORs/nucleus were present, whereas of grade III tumours 8/18 (44%) showed usually more than 3.5 Ag-NORs/nucleus (p = 0.0163). The number of Ag-NORs was significantly related to mean nuclear area (NA) (p = 0.017) and to SD of nuclear area (p = 0.05). The Ag-NORs were not related significantly to clinical stage, perineural infiltration, lymphatic infiltration, DNA ploidy, SPF, G2 fraction or M/V index, although there was a clear trend between the variables. In survival analysis, the degree of lymphatic infiltration (LI) (P = 0.009) predicted survival, whereas AgNORs had no significant prognostic value albeit a trend was observed. In T1-T2 tumours, histological grade (p = 0.05), PNI (p = 0.04) and SPF (p = 0.0076) predicted survival.     

Prognostic value of cell proliferation in breast cancer as determined by proliferating cell nuclear antigen (PCNA) immunostaining.

A series of 175 biopsies from primary tumours were subjected to immunostaining for proliferation cell nuclear antigen (PCNA) and histopathological analysis for prognostic factors in 175 women with breast cancer followed up for nine years. In normal breast epithelium, only occasional nuclei were positive for PCNA. In breast carcinomas, the fraction of nuclei positive for PCNA showed considerable intratumoural variation, usually being highest at the invasive tumour margins. The fraction of positive nuclei was significantly related to histological grade (p less than 0.001), histological type (p = 0.049) and tumor recurrence (p = 0.01). The fraction of positive nuclei predicted recurrence-free survival (p = 0.007) and overall survival (p = 0.0158) in univariate analysis. In the multivariate analysis including also the standard prognostic factors, the PCNA positivity predicted recurrence-free survival (p = 0.004) and overall survival (p = 0.030) independently. The results show that the light microscopic assessment of the growth fraction as determined by PCNA immunolabeling has independent prognostic value in breast carcinomas like some other (e.g. S phase fraction, mitotic index, Ki-67) previously characterized proliferation indices.     

Lack of effect of hormonal treatment and smoking on cytokeratin expression in buccal mucosa

Differences in cytokeratin expression of clinically normal buccal mucosa were studied in 50 healthy women by indirect immunofluorescence staining with monoclonal antibodies. The subjects were divided into four groups: control group (N= 18), smokers (N=8), oral contraceptive users (N=8) and smokers receiving oral contraceptives (N=16). Our findings indicate that cytokeratin expression in non-cornified stratified epithelium is not influenced by smoking or oestradiol/progesterone treatment. Only cytokeratin No. 19 showed variable patterns of expression but the differences could not be ascribed to smoking or contraceptives. Cytokeratin No. 19 gave a positive reaction in the basal and suprabasal layers in 34 subjects (68%). In 9 (18%) specimens, the staining was positive in the basal cells and showed a positive heterogeneous cytoplasmic reaction in the suprabasal cells. Interestingly, cytokeratin No. 7 was recognized in all epithelial cells except the basal cells. Our results suggest that changes in the serum oestradiol levels do not affect the cytokeratin pattern in buccal mucosa.     

Intrapelvic cyst formation after hip arthroplasty with a carbon fibre-reinforced polyethylene socket

Carbon fibre-reinforced polyethylene cups were inserted in total hip arthroplasties for both hips of a male patient. Ten years after the arthroplasty of the right hip, an intrapelvic cyst was observed. The cyst was connected to the inner acetabular wall. The endoprosthetic cup showed gross loosening with a marked osteolysis of the acetabulum. Revision arthroplasty with allograft bone and an uncemented endoprosthesis was successfully performed. The fibrotic tissue at the bone-cement interface showed numerous histiocytic cells with cytoplasmic infiltrates of carbon and polyethylene particles. The left hip was also later revised for loosening, but no cyst formation was observed on that side.     

Bronchial carcinoma and its regional lymph nodes in relation to immunological functions.

Two hundred and fifty lymph nodes from 100 patients with lung carcinoma and the same number of regional nodes from 119 patients with gastric ulcer were histologically evaluated by the standardized reporting system of COTTIER et al. Special attention was focused on the immunological reactions in these lymph nodes. Histologic features suggesting actively functioning humoral and cell-mediated immune reactions were encountered more frequently in the nodes of the control series. The significance of the histologic findings is discussed and it is concluded that there is some degree of derangement of both humoral and cell-mediated immune responses in the lymph nodes draining lung carcinoma. The applicability of the reporting system used is emphasized.     

Psoriasin (S100A7) and calgranulin-B (S100A9) induction is dependent on reactive oxygen species and is downregulated by Bcl-2 and antioxidants

S-100 proteins are calcium-binding proteins with important growth regulatory functions. Of these proteins, psoriasin and calgranulin-B have been shown to be highly upregulated in ductal carcinoma in situ (DCIS) of the breast and in psoriasis. The purpose of this study was to further elucidate the functional relevance of the overexpression of these two S-100 proteins in psoriasis and DCIS. We report the induction of both proteins by reactive oxygen species, phorbol ester TPA, and the induction of psoriasin in response to the PI3K inhibitor wortmannin. We also demonstrate that Bcl-2 overexpression represses the induction of psoriasin and calgranulin-B under these different conditions. The same effect was obtained with the antioxidant NAC, which indicates that the suppression of psoriasin and calgranulin-B induction is mediated by the antioxidant function of Bcl-2. Furthermore, we demonstrate that overexpression of a dominant negative IKKbeta also inhibits the induction of psoriasin suggesting that the NFkappaB pathway is involved in the induction of this protein. Also, we found NFkappaB responsive DNA elements in the upstream promoter region of psoriasin. MCF10A cells with a stable retroviral overexpression of psoriasin were significantly more resistant to H2O2-induced cell death than control cells further supporting the hypothesis that these S-100 proteins may play a role in oxidative stress response.     

Human papilloma virus (HPV) antigens in lesions of laryngeal squamous cell carcinomas

Paraffin sections from 36 laryngeal squamous cell carcinomas with histological features of coexistent condylomatous changes (the flat, inverted and papillomatous lesions caused by human papilloma virus; HPV) were stained for HPV antigens with an indirect immunoperoxidase technique. The HPV antiserum used was prepared in guinea pigs immunized against the purified HPV virions from human common wart tissue. Of the tumors studied, 36% showed the presence of HPV antigens. In that, one-third of the flat and inverted lesions and 42% of the papillomatous lesions contained cells with intranuclear HPV-staining reaction. The role of HPV as the etiologic agent of laryngeal squamous cell papillomas is discussed, as is the potential malignant transformation of them. Based on the present results, the previously expressed view on the probable role of HPV in the development of laryngeal squamous cell carcinoma seems to gain further substantiation.