Processing, secretion, and biological properties of a novel growth factor of the fibroblast growth factor family with oncogenic potential.

We recently reported that the protein encoded in a novel human oncogene isolated from Kaposi sarcoma DNA was a growth factor with significant homology to basic and acidic fibroblast growth factors (FGFs). To study the properties of this growth factor (referred to as K-FGF) and the mechanism by which the K-fgf oncogene transforms cells, we have studied the production and processing of K-FGF in COS-1 cells transfected with a plasmid encoding the K-fgf cDNA. The results show that, unlike basic and acidic FGFs, the K-FGF protein is cleaved after a signal peptide, glycosylated, and efficiently secreted as a mature protein of 176 or 175 amino acids. Inhibition of glycosylation impaired secretion, and the stability of the secreted K-FGF was greatly enhanced by the presence of heparin in the cultured medium. We have used the conditioned medium from transfected COS-1 cells to test K-FGF biological activity. Similar to basic FGF, the K-FGF protein was mitogenic for fibroblasts and endothelial cells and induced the growth of NIH 3T3 mouse cells in serum-free medium. Accordingly, K-fgf-transformed NIH 3T3 cells grew in serum-free medium, consistent with an autocrine mechanism of growth. We have also expressed the protein encoded in the K-fgf protooncogene in COS-1 cells, and it was indistinguishable in its molecular weight, glycosylation, secretion, and biological activity from K-FGF. Taken together, these results suggest that the mechanism of activation of this oncogene is due to overexpression rather than to mutations in the coding sequences.

Presence of chromosomal abnormalities and lack of AIDS retrovirus DNA sequences in AIDS-associated Kaposi's sarcoma.

The frequent occurrence of Kaposi's sarcoma (KS) in association with the acquired immune deficiency syndrome (AIDS) could be due to the fact that the etiological agent of this tumor is the same retrovirus causing AIDS, to another oncogenic virus frequently found in AIDS patients, or to the unmasking of the tumorigenic potential of KS cells by immunosuppression. We have therefore investigated the presence of DNA sequences homologous to the AIDS retrovirus, cytomegalovirus (CMV), and hepatitis B virus in 13 KS necropsies and biopsies from AIDS patients. All KS DNA samples were negative for AIDS retrovirus or hepatitis B DNA sequences. Two DNAs from necropsies contained CMV DNA, but the data suggested the presence of replicating CMV DNA due to generalized infection. We have also studied cell cultures derived from KS skin biopsies of AIDS patients. These cultures had a short lifetime in vitro and expressed some markers of endothelial cells. The cells were not tumorigenic in nude mice but contained a number of chromosomal rearrangements which were often monoclonal within the same culture. However, these abnormalities were different from culture to culture and even in cultures from the same biopsy. The presence of these chromosomal abnormalities seemed to correlate with the cell positivity for endothelial markers. Taken together these results indicate that neither the AIDS retrovirus, CMV, or hepatitis B virus is directly responsible for the altered growth of KS cells, that KS may be polyclonal even within the same lesion, and that KS cells have a tendency to karyotypic rearrangements.

[Neurologic and cerebrovascular pathology during treatment with oral contraceptives]. / Patologia vascolare encefalica e neurologica in corso di trattamento con contraccettivi orali.

PIP: Between 1970-1978 a total of 3146 women aged 12-50 were hospitalized in the neurologic division of the Regional Hospital of Borgo Trento, Italy. Of these, only 47 patients, average age 27.8, were on oral contraceptives (OCs); average hospitalization time was of 9.7 days. Main complaints were headache, nausea, vomiting, depression, and vertigo. However, only 12 of 47 patients presented with symptoms: 5 cases, or 41.6%, of anomalous encephalogram, and 7 cases, or 58.4%, of cerebrovascular problems. These clinical manifestations are often reported in other studies. The article contains an extensive review of the medical literature published on the subject.^ieng

Effect of median-nerve electrical stimulation on BOLD activity in acute ischemic stroke patients.

OBJECTIVE: To investigate blood oxygenation level-dependent (BOLD) activation during somatosensory electrical stimulation of the median nerve in acute stroke patients and to determine its correlation with ischemic damage and clinical recovery over time. METHODS: Fourteen acute stroke patients underwent functional magnetic resonance imaging (fMRI) during contralesional median-nerve electrical stimulation 12-48 h after stroke. Findings were then validated by diffusion tensor imaging (DTI) and motor evoked potential by transcranial magnetic stimulation (TMS). RESULTS: Poor clinical recovery at three months was noted in four patients with no activation in the early days after stroke, whereas good clinical recovery was observed in eight patients with a normal activation pattern in the primary sensory motor area in the acute phase. In two patients BOLD activation correlated weakly with clinical recovery. Findings from TMS and DTI partially correlated with clinical recovery and functional scores. CONCLUSIONS: Clinically relevant insights into the "functional reserve" of stroke patients gained with peripheral nerve stimulation during fMRI may carry prognostic value already in the acute period of a cerebrovascular accident. SIGNIFICANCE: BOLD activation maps could provide insights into the functional organization of the residual systems and could contribute to medical decision making in neurological and rehabilitative treatment.

Are indexes of arm recovery related to daily life autonomy in patients with stroke?

AIM: The level of daily life autonomy in patients with stroke may be related to recovery of affected arm function. The aim of the study was to assess whether four simple bedside indexes of arm recovery can predict levels of autonomy in daily life activities. METHODS: A consecutive sample of 48 patients presenting with upper limb paresis/plegia in the acute stage after stroke was selected. Patients underwent five evaluation sessions at 7, 14, 30, 90 and 180 days after stroke. Forward stepwise multiple regression analysis was used to clarify the role of four potential predictors of upper limb recovery (active finger extension, shoulder abduction, shoulder shrug and hand movement scales). Dependent variables in these models were the Barthel Index score and sub-items of the Burke-Fahn-Marsden Scale. RESULTS: The active finger extension scale showed a highly significant statistical correlation with patient performance in nearly all outcome measures. The shoulder shrug correlated with the BI score, and with the dressing and hygiene Burke-Fahn-Marsden Scale sub-items. Shoulder abduction and hand movement scale played only a minor role. CONCLUSIONS: The active finger extension scale proved to be a strong early predictor of recovery of daily life autonomy in patients with stroke. This finding could be important in order to planning a specific rehabilitation treatment after the onset.

Dural arteriovenous fistulas with aggressive course: clinical and angiographic correlations in two patients.

Cranial dural arteriovenous fistulas (DAVFs) usually present with non-aggressive symptoms. We here report two patients who presented a peculiar clinical picture related to DAVFs, with focal neurological signs and haemorrhagic (case 1) or ischaemic lesions (case 2) respectively. The clinical and angiographic findings and putative pathophysiological mechanisms are discussed.

Isolation of a rearranged human transforming gene following transfection of Kaposi sarcoma DNA.

By transfecting high molecular weight DNA from a Kaposi sarcoma lesion into murine NIH 3T3 cells, we have identified and molecularly cloned a set of human DNA sequences capable of inducing focus formation, growth in agar, and tumorigenicity in these cells. The human DNA sequences present in primary, secondary, and tertiary NIH 3T3 transformants encompass about 32 kilobases (kb) and contain four rearrangements with respect to normal human DNA and a portion of the c-fms protooncogene (FMS in human gene nomenclature). However, the minimal transforming region (6.6 kb) identified in our cloned DNA borders on the c-fms DNA region but does not contain c-fms coding sequences. The fms sequences are also not represented in the two transcripts (approximately equal to 1.2 and 3.5 kb) detected in NIH 3T3 transformants; however, they might provide elements regulating expression. Hybridization to several known oncogene probes and preliminary sequencing data indicate that we have identified a previously unrecognized "activated" oncogene. Since the rearrangements present in our cloned DNA sequences are not detectable in the original Kaposi tumor DNA used for transfection, it is possible that this oncogene was generated during gene transfer.

A reiterated leader sequence is present in polyomavirus late transcripts produced by a transformed rat cell line.

In cells transformed by polyomavirus, the viral genome is integrated into the host DNA, and in the absence of excision, viral gene expression is limited to the early region. We report here that the ability of a unique transformed rat cell line, designated SS1A, to produce readily detectable levels of late mRNAs is due to rearrangements of the integrated viral sequences. The structure of the SS1A insertion, resulting from amplification and deletion events, allows for the formation of a primary late transcript that can subsequently be spliced to generate a reiterated leader attached to the body of the late mRNA coding sequences. The presence of transcripts containing such a leader was confirmed by sequencing the 5' end of cDNA copies of late mRNAs isolated from a library constructed with SS1A mRNA. These results suggest that a reiterated leader sequence is necessary to stabilize late mRNA.

Ultrastructural observations of polyoma infected Friend erythroleukemic cells.

Friend erythroleukemic cells (FLC) infected by Polyoma (Py) virus were studied at the electron microscope both during their lytic cycle, which occurs within the first 96 hr after infection, and in the surviving stabilized cells 30 days after infection. The results showed that the Polyoma virus and Friend leukemic virus (FLV) coexist and mature together in the same cell. During the lytic cycle, the Py particles were scattered in the nucleoplasm, and sometimes they were also found aggregated into crystals. Instead crystalline aggregates of Py particles were always present in the few surviving virus-carrying cells 30 days after infection. A possible interpretation could be that the crystal aggregation serves to maintain the viruses in a latency that allows the survival of the host cells.

bFGF as an autocrine growth factor for human melanomas.

Normal human melanocytes in culture require specific additives such as basic fibroblast growth factor (bFGF) and dibutyryl cyclic adenosine monophosphate (dbcAMP) in order to proliferate in defined or serum-containing medium (Halaban et al., 1987). This stringent requirement is absent in cells derived from metastatic melanomas which not only proliferate in regular culture medium, but also produce a substance immunologically related to bFGF (Halaban et al., 1987). We show here that the mitogenic activity necessary for normal human melanocytes is constitutively present in several lines of human metastatic melanomas and that this activity is inactivated by anti-bFGF antibodies. Melanoma cells, but not normal melanocytes, express bFGF gene transcripts. Although the molecular mechanism underlying the abnormal expression of bFGF in melanomas is not known, the results suggest that bFGF acts as an autocrine growth factor in melanomas.

The FGF-related oncogene, K-FGF, maps to human chromosome region 11q13, possibly near int-2.

The protein encoded in a novel human oncogene isolated by transfection of Kaposi's sarcoma DNA is a growth factor with significant homology to basic and acidic FGFs. The genomic structure of this oncogene (designated K-FGF), as originally isolated, carried DNA rearrangements upstream and downstream of the coding region. The normally discontinuous sequence upstream of the K-FGF coding region derived from the 3' end of the c-fms gene and thus originated from human chromosome 5. In order to determine the normal chromosomal location of the K-FGF gene and of the DNA sequences adjacent to its 3' end, we have correlated the presence of these sequences with retention of specific human chromosome regions in rodent-human somatic cell hybrids. These experiments mapped the K-FGF gene to human chromosome region 11q13----11q23, and in situ hybridization localized it more precisely to region 11q13 near int-2, which also belongs to the FGF family. The sequence downstream of the gene in transfectants and discontinuous with K-FGF in normal human DNA derives from chromosome region 12p12----12q13, possibly near the int-1 locus.

Polyomavirus growth and persistence in Friend erythroleukemic cells.

Infection of Friend erythroleukemic (FL) cells by polyomavirus (Py) invariably results in the selection of persistently infected FL-Py cell lines and clones. Anti-Py serum treatment of FL-Py lines and clones leads to the loss of Py genome and consequent cell cure. Conversely, cure has not been obtained in FL-PytsA cell lines (isolated after infection by a Py thermosensitive early mutant) and their derivative clones cultivated for a long time at nonpermissive temperature (39 degrees C), where viral large-T protein is inactive. Rescue of viral particles has always been obtained after shifting cells to 32 degrees C. Integrated viral genomes were detected by blot hybridization in an FL-PytsA clone at 39 degrees C. Long-term observation of FL-Py cell lines and their derivative clones reveals a reciprocal selection mechanism (coevolution) between the viral and the cellular populations, resulting in either a completely virus-free Py-resistant FL cell line (cure) or in a continuously Py-shedding line bearing Py genome variants. Structural analysis of these viral populations has been carried out, and some viral variants have been isolated and characterized. On the basis of the results obtained, the possible mechanisms of Py persistence in FL cells will be discussed.

Mitogenic and dedifferentiating effect of the K-fgf/hst oncogene on rat thyroid PC clone 3 epithelial cells.

Transfection of rat thyroid differentiated epithelial cells, PC clone 3, with the K-fgf/hst oncogene results in alleviation of thyrotropin dependency for growth and suppression of the differentiated phenotype without the acquisition of the fully transformed phenotype. An autocrine mechanism is responsible for these effects, since the proliferation of PC clone 3 cells, induced by K-FGF-conditioned medium, is blocked by anti-K-FGF-specific antibodies. Moreover, addition of K-FGF-conditioned medium inhibits the thyroid differentiated functions. Also, such other members of the fibroblast growth factor family as basic and acidic fibroblast growth factor are able to induce thyroid cell growth and to block expression of the differentiated functions.

An oncogene isolated by transfection of Kaposi's sarcoma DNA encodes a growth factor that is a member of the FGF family.

We recently reported the cloning of a rearranged human oncogene following transfection of DNA from Kaposi's sarcoma into NIH 3T3 cells. To identify the protein(s) encoded in two novel mRNAs of 3.5 and 1.2 kb expressed in NIH 3T3 transformants, we constructed a cDNA library. One of the cDNA clones isolated (KS3) corresponded to the 1.2 kb mRNA and transformed NIH 3T3 cell when inserted into a mammalian expression vector. The 1152 nucleotide KS3 cDNA encodes a protein of 206 amino acids with significant homology to the growth factors basic FGF and acidic FGF. Expression of the KS3 product as a bacterial fusion protein or in COS cells allowed us to determine that both proteins had significant growth-promoting activity and that the COS cell protein was glycosylated. Thus one of the mRNAs transcribed from the KS oncogene encodes a growth factor that could transform cells by an autocrine mechanism and appears to represent a new member of the FGF family.